Functionalization of endogenous bacteria

ABSTRACT

Various aspects and embodiments of the present disclosure are directed to methods and compositions for functionalizing endogenous bacteria in vivo. The methods include delivering to endogenous bacterial cells a recombinant bacteriophage or phagemid that is engineered to contain at least one genetic circuit.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 15/924,045, filed Mar. 16, 2018, which is a continuation of U.S. application Ser. No. 14/320,965, filed Jul. 1, 2014, now U.S. Pat. No. 9,957,511, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 61/841,904, filed Jul. 1, 2013, each of which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. DP2 OD008435 and T32 GM008334 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

A human microbiome is considered to be the aggregate of microorganisms that reside on the surface and in deep layers of the skin, in the saliva and oral mucosa, in the conjunctive, an in the gastrointestinal tracts. A human microbiome is thought to be composed of at least ten times as many bacterial cells as human cells, and these bacterial communities are known to have major impacts on the systems in which they reside. For example, recent studies have implicated the human microbiome in many human diseases.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the surprising discovery that endogenous bacterial cells of a microbiome (e.g., human microbiome) can be “functionalized” in vivo with new genetically encoded capabilities to perform a range of useful functions. Thus, the present disclosure provides, inter alia, methods and compositions for selectively manipulating bacterial cells that are stably maintained in a microbiome (e.g., endogenous bacterial cells), for example, to deliver therapeutic molecules and/or to serve as “biosensors.” The present disclosure is also based on the discovery that recombinant bacteriophages (e.g., non-lytic, or lysogenic bacteriophages) can be engineered to selectively deliver, to endogenous bacterial cells in vivo, particular genetic circuits, which when expressed, functionalize the bacterial cells.

Thus, various aspects and embodiments of the present disclosure provide methods of functionalizing endogenous bacteria in vivo, the methods comprising delivering to endogenous bacterial cells a recombinant bacteriophage that is engineered to contain at least one genetic circuit. Other aspects and embodiments of the present disclosure contemplate the delivery of recombinant phagemids to endogenous bacterial cells.

In some embodiments, the at least one genetic circuit does not express an antimicrobial protein or peptide.

Various other aspects and embodiments of the present disclosure provide recombinant bacteriophages that are engineered for in vivo delivery to endogenous bacterial cells and to contain at least one genetic circuit, wherein the at least one genetic circuit does not express an antimicrobial protein or peptide. Other aspects and embodiments of the present disclosure provide recombinant phagemids that are engineered for in vivo delivery to endogenous bacterial cells and to contain at least one genetic circuit, wherein the at least one genetic circuit does not express an antimicrobial protein.

In some embodiments, the endogenous bacterial cells are nonpathogenic bacterial cells.

In some embodiments, the endogenous bacterial cells are stably maintained in a microbiome.

In some embodiments, the recombinant bacteriophage is a non-lytic recombinant bacteriophage. In some embodiments, non-lytic bacteriophages, or phagemids, are from a family selected from Myoviridae, Siphoviridae, Podoviridae, Tectiviridae, Corticoviridae, Lipothrixviridae, Plasmaviridae, Rudiviridae, Fuselloviridae, Inoviridae, Microviridae, Leviviridae and Cystoviridae. In some embodiments, wherein a non-lytic recombinant Inoviridae bacteriophage is an M13 or M13-like bacteriophage. In some embodiments, a recombinant phagemid is an M13-derived phagemid.

In some embodiments, the genetic circuit contains a nucleic acid with a promoter operably linked to a nucleotide sequence encoding a gene product.

In some embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, the nucleic acid is a synthetic nucleic acid.

In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is an inducible promoter.

In some embodiments, the genetic circuit is a recombinase-based genetic circuit. In some embodiments, the genetic circuit is engineered to express a therapeutic molecule. In some embodiments, the genetic circuit comprises a nucleic acid with a promoter operably linked to a nucleotide sequence that encodes the therapeutic molecule. In some embodiments, the therapeutic molecule is an antibody, antibody-based drug, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin or thrombolytic.

In some embodiments, the genetic circuit is engineered to detect a condition. In some embodiments, the condition is a cancer, an immune disorder or an infection.

In some embodiments, the genetic circuit comprises a nucleic acid with an inducible promoter operably linked to a nucleotide sequence that encodes a reporter molecule. In some embodiments, the reporter molecule is a fluorescent protein.

In some embodiments, the genetic circuit comprises a nucleic acid with a promoter operably linked to a nucleotide sequence that encodes toluene dioxygenase or styrene monoxygenase.

Also provided herein are compositions that comprise any one or more of the recombinant bacteriophages and/or phagemids of the present disclosure.

As used herein, “a” and “an” should be understood to mean “at least one.”

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing.

FIG. 1 shows a graph of indigo concentration over time for the constitutive PLtet0 and the inducible Pbad promoter networks used to express toluene dioxygenase. Toluene dioxygenase (and, thus, indigo) is produced in the presence of the Pbad inducer and in the presence of the Ptet inducer.

FIG. 2 shows a graph of indigo concentration over time for the constitutive PLtet0 and the inducible Pbad promoter networks used to express styrene monooxygenase. Styrene monooxygenase (and, thus, indigo) is produced in the presence of the Pbad inducer and in the presence of the Ptet inducer.

FIG. 3 depicts a graph of indigo measurements after overnight infection of bacteria with recombinant M13 bacteriophage harboring different plasmids.

FIG. 4 shows images of plates from an overnight spot test of M13-pBAD-029, M13-kan-029 and M13-pTet-030. Image on the left is with a black backdrop; image on the right is with a white backdrop. For each plate, the spots from top to bottom are: M13-gfp, M13-mCherry, M13-pBAD-styAB, M13-kan-pBAD-styAB, and M13-pTet-styAB. Spots represent a 1:10 dilution series from right to left, starting with 7.5 μL of stock.

FIG. 5 shows a graph of indigo concentration over time produced by bacteria transformed with the inducible Pbad promoter network expressing styrene monooxygenase (“test network”) or infected with recombinant M13 bacteriophage harboring the inducible Pbad promoter network expressing styrene monooxygenase (“bacteriophage network”) (top). FIG. 5 also shows a graph of indigo concentration over time produced by bacteria transformed with the constitutive PLtet0 promoter network expressing styrene monooxygenase (“test network”) or infected with recombinant M13 bacteriophage harboring the constitutive PLtet0 promoter network expressing styrene monooxygenase (“bacteriophage network”) (bottom). Styrene monooxygenase (and, thus, indigo) is produced in the presence of the Pbad inducer for both the test network and the bacteriophage network.

FIG. 6 shows a graph of the fold change of the concentration of indigo produced over time by bacteria transformed with the inducible Pbad test network versus bacteria infected with the inducible Pbad bacteriophage network (top). FIG. 6 also shows a graph of the fold change of the concentration of indigo produced over time by bacteria transformed with the constitutive PLtet0 test network versus bacteria infected with the constitutive PLtet0 bacteriophage network (bottom).

FIG. 7 shows a graph of indigo concentration as a function of arabinose inducer dosage. EMG2 cells were infected with M13 bacteriophage carrying the inducible Pbad promoter network expressing styrene monooxygenase (M029).

FIG. 8A depicts a schematic of a general in vivo model of the present disclosure.

FIG. 8B depicts a general in vivo Inflammatory Bowel Disease model of the present disclosure.

FIG. 9 depicts an inducible version of toluene dioxygenase on a test plasmid (pRJK025).

FIG. 10 depicts a constitutive version of toluene dioxygenase on a test plasmid (pRJK026).

FIG. 11 depicts an inducible version of styrene monooxygenase on a test plasmid (pRJK029).

FIG. 12 depicts a constitutive version of styrene monooxygenase on a test plasmid (pRJK030).

FIG. 13 depicts an inducible version of styrene monooxygenase on an M13 plasmid (M029).

FIG. 14 depicts a constitutive version of styrene monooxygenase on an M13 plasmid (M030).

FIG. 15A depicts an example of a genetic circuit of the present disclosure, which includes a P_(TRC-2) promoter operably linked to a ribosomal binding site and a nucleotide sequence encoding green fluorescent protein (GFP) with a LAA protein tag. FIG. 15B depicts a graph showing the change in GFP fluorescence over time obtained from (1) ER2738 Escherichia coli (E. coli) cells transformed with a bacteriophage engineered to contain the genetic circuit depicted in FIG. 15A (referred to as a M6G bacteriophage), (2) ER2738 E. coli cells transformed with a phagemid engineered to contain the genetic circuit depicted in FIG. 15A (referred to as a p6T phagemid), and (3) ER2738 E. coli negative control cells (bacteria only).

FIG. 16 depicts a graph showing the percentage of GFP positive cells in gnotobiotic mice colonized with (1) M6G bacteriophages, (2) ER2738 E. coli cells transformed with pLT006 plasmids containing the genetic circuit depicted in FIG. 15A (positive control), and (3) ER2738 E. coli negative control cells.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are efficient and effective strategies for selectively manipulating endogenous bacterial cells of a microbiome to express genetic circuits, for example, to deliver therapeutic molecules to the host organism or to detect changes in the host organism's biological environment. Surprisingly, the present disclosure shows that these endogenous bacterial cells, which are stably maintained in a host organism, can be manipulated in vivo through the use of viruses (e.g., bacteriophages), or virus particles (e.g., phagemids), to serve as, inter alia, long-term drug delivery devices and biosensors. One traditional way of manipulating a microbiome is to administer antibiotics; however, antibiotics are broad spectrum in nature and target/kill both pathogenic and non-pathogenic “healthy” bacteria. Another traditional way of manipulating a microbiome is to deliver exogenous bacteria in the form of, for example, probiotic pills or yogurt; however, exogenous bacteria are typically only transiently present in the microbiome to which they are delivered because most existing ecological niches are already occupied by endogenous bacteria. Unlike the foregoing traditional strategies, which are indiscriminate and transient in nature, the present disclosure provides, in some instances, selective and stable delivery of new genetic programs to endogenous bacterial cells already established in a microbiome.

A “microbiome,” as used herein, refers to the totality of microbes in a particular environment (e.g., in/on an organism, in a marine environment (e.g., ocean), and/or in a terrestrial environment (e.g., soil)). In some embodiments, a microbiome may refer to the totality of microbes that reside, or are stably maintained, for example, on the surface and in deep layers of the skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tracts of an organism. A “host” organism or subject (e.g., animal such as a mammal, e.g., human) refers to the organism in/on which endogenous bacteria reside. Examples of host organisms or subjects in accordance with the present disclosure include, without limitation, animals such as humans, domesticated animals (e.g., cats, dogs, rodents, rabbits, birds), and farm animals (e.g., cows, pigs, goats, chickens, horse, sheep). It is to be understood that the microbes (e.g., endogenous bacteria) of a microbiome are typically non-pathogenic, or “healthy,” microbes (e.g., they exist symbiotically, or in a mutually beneficial relationship, in an organism and do not cause disease unless the microbes grow abnormally). The bacterial cells (e.g., non-pathogenic bacterial cells) that make up a microbiome are referred to herein as “endogenous” bacterial cells. The endogenous bacteria that exist in/on an organism are distinguished from exogenous bacteria, which can be introduced to an organism and, in some instances, may be pathogenic (e.g., may cause disease).

The present disclosure is directed, in some embodiments, to methods of functionalizing endogenous bacteria in vivo to express genetic circuits that can be used, for example, to deliver therapeutic molecules and/or to act as biosensors of various biological conditions and/or disease states. To achieve this functionalization, the present disclosure contemplates, in some embodiments, engineering non-lytic, or lysogenic, recombinant bacteriophages and/or phagemids as vehicles to deliver to endogenous bacterial cells a variety of new genetic programs.

Bacteriophages

A bacteriophage (also referred to as a phage), is a virus that infects and replicates in bacteria. Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Bacteriophages replicate within bacteria following the injection of their genome into the cytoplasm and do so using either a lytic cycle, which results in bacterial cell lysis, or a lysogenic (non-lytic) cycle, which leaves the bacterial cell intact. Bacteriophages of the present disclosure are, in some embodiments, non-lytic (also referred to as lysogenic or temperate). Thus, after phage delivery of a genetic circuit to an endogenous bacterial cell, the bacterial cell may remain viable and able to stably maintain expression of the genetic circuit.

Examples of non-lytic bacteriophage for use in accordance with the present disclosure include, without limitation, Myoviridae (P1-like viruses; P2-like viruses; Mu-like viruses; SPO1-like viruses; phiH-like viruses); Siphoviridae (k-like viruses, γ-like viruses, T1-like viruses; T5-like viruses; c2-like viruses; L5-like viruses; .psi.M1-like viruses; phiC31-like viruses; N15-like viruses); Podoviridae (phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus). Such phages may be naturally occurring or engineered phages. In some embodiments, a bacteriophage is a coliphage (e.g., infects Escherichia coli).

In some embodiments, a bacteriophage of the present disclosure is an M13 bacteriophage. M13 is a filamentous bacteriophage of the family Inoviridae and is composed of circular single-stranded DNA. M13 phages are about 900 nm long and 6-7 nm in diameter with 5 proteins. The minor coat protein, P3, attaches to the receptor at the tip of the F pilus of an Escherichia coli host cell. Thus, in some embodiments, methods of the present disclosure comprise delivering to endogenous bacterial cells a recombinant M13 bacteriophage that is engineered to contain at least one genetic circuit.

In some embodiments, the bacteriophage of the present disclosure is isolated from (e.g., collected from, obtained from) stool or sewage.

Phagemids

In some embodiments of the present disclosure, phagemids are engineered to contain at least one genetic circuit. As used herein, “phagemid” refers to a bacteriophage-derived vector containing the replication origin of a plasmid and the packaging site of a bacteriophage. Examples of phagemids that may be used in accordance with the present disclosure include, without limitation, M13-derived phagemids containing the f1 origin for filamentous bacteriophage packaging such as, for example, pBluescript II SK (+/−) and KS (+/−) phagemids, pBC SK and KS phagemids, pADL and P1-based phagemids (see, e.g., Westwater C A et al., Microbiology 148, 943-50 (2002); Kittleson J T et al., ACS Synthetic Biology 1, 583-89 (2012); Mead D A et al., Biotechnology 10, 85-102 (1988)). Other phagemids may be used in accordance with the present disclosure and, for example, can be made to work with packaging systems from natural, engineered or evolved bacteriophage.

Endogenous Bacterial Cells

A microbiome may comprise of a variety of endogenous bacterial species, any of which may be targeted in accordance with the present disclosure. In some embodiments, the genus and/or species of target endogenous bacterial cells may depend on the type of bacteriophage and/or phagemid being used. For example, some bacteriophages exhibit tropism for, or preferentially target, specific host species of bacteria. Other bacteriophages do not exhibit such tropism and may be used to target a number of different genus and/or species of endogenous bacterial cells. In some embodiments, the bacteria are considered to be lysogenic bacteria. As used herein, “lysogenic bacteria” are endogenous bacteria that are infected by a non-lytic (also referred to as lysogenic or temperate) bacteriophage. Lysogenic bacteria are typically not lysed by bacteriophage infection. It should be appreciated that, in some instances, infection of an endogenous bacterial cell by a non-lytic or lysogenic bacteriophage may result in cell lysis of, for example, a small proportion (e.g., less than 20%, less than 10%, less than 5%, less than 2%, or less than 1%) of the infected bacterial cells.

Bacteria are small (typical linear dimensions of around 1 micron), non-compartmentalized, with circular DNA and ribosomes of 70S. As used herein, the term “bacteria” encompasses all variants of endogenous bacteria. “Endogenous” bacteria naturally reside in a closed system. Bacterial cells of the present disclosure include bacterial subdivisions of Eubacteria and Archaebacteria. Eubacteria can be further subdivided into gram-positive and gram-negative Eubacteria, which depend upon a difference in cell wall structure. Also included herein are those classified based on gross morphology alone (e.g., cocci, bacilli). In some embodiments, the bacterial cells are Gram-negative cells, and in some embodiments, the bacterial cells are Gram-positive cells. Examples of bacterial cells of the present disclosure include, without limitation, cells from Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp., Bacteroides spp., Prevotella spp., Clostridium spp., Bifidobacterium spp., or Lactobacillus spp. In some embodiments, the bacterial cells are from Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Actinobacillus actinobycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphylococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Streptococcus Enterococcus faecalis, Bacillus coagulans, Bacillus ceretus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Zymomonas mobilis, Streptomyces phaechromogenes, or Streptomyces ghanaenis. Thus, bacteriophages and/or phagemids of the present disclosure may target (e.g., specifically target) a bacterial cell from any one or more of the foregoing genus and/or species of bacteria.

In some embodiments, bacterial cells of the present disclosure are anaerobic bacterial cells (e.g., cells that do not require oxygen for growth). Anaerobic bacterial cells include facultative anaerobic cells such as, for example, Escherichia coli, Shewanella oneidensis and Listeria. Anaerobic bacterial cells also include obligate anaerobic cells such as, for example, Bacteroides and Clostridium species. In humans, for example, anaerobic bacteria are most commonly found in the gastrointestinal tract. Thus, bacteriophages and/or phagemids of the present disclosure may target (e.g., specifically target) anaerobic bacterial cells.

Genetic Circuits

The non-lytic recombinant bacteriophages and/or phagemid of the present disclosure are engineered to deliver to endogenous bacterial cells one or more of a variety of genetic circuits, which may depend on the particular intended application (e.g., delivery of a therapeutic molecule). A “genetic circuit,” as used herein, refers to a nucleic acid containing at least one promoter operably linked to at least one nucleic acid sequence that encodes a gene product of interest (e.g., protein or RNA). In some embodiments, a genetic circuit contains a nucleic acid with a promoter operably linked to a single gene, and in other embodiments, the promoter is operably linked to a cluster of genes (e.g., an operon). In yet other embodiments, a genetic circuit may contain more than one promoter, each linked (e.g., operably linked) to the same gene, the same cluster of genes, or to a different gene or cluster of genes. Herein, a genetic circuit that is “engineered to express” a particular gene product (e.g., a therapeutic molecule) may be a genetic circuit that contains at least the genetic elements required for expression of the particular gene product. For example, a genetic circuit that is engineered to express a monoclonal antibody may contain at least one nucleic acid with at least one promoter operably linked to at least one nucleotide sequence encoding the primary amino acid sequence of the monoclonal antibody. Herein, a genetic circuit that is “engineered to detect” a particular condition (e.g., a disease state) may be a genetic circuit that contains at least the genetic elements required for regulated expression of a reporter molecule or other molecule that can be visualized or quantified. For example, a genetic circuit that is engineered to detect oxidative stress via pOxyS or pSoxS and to respond by expressing a styrene monoxygenase enzyme or an anti-inflammatory gene such as, for example, interleukin 10. Styrene monooxygenase converts indole, present in the gut, to indigo as a visual readout.

The genetic circuits of the present disclosure may be constitutive or regulated. A “constitutive” genetic circuit, as used herein, is active in all circumstances in the bacterial cell, whereas a “regulated” genetic circuit becomes active, or inactive, in response to specific stimuli. A genetic circuit is considered to be “active” when a promoter, described elsewhere herein, initiates transcription of an operably linked nucleotide sequence (e.g., gene sequence). A genetic circuit is considered to be “inactive” when transcription of an operably linked nucleotide sequence is terminated. A constitutive genetic circuit may contain a nucleic acid with a constitutive promoter operably linked to nucleotide sequence encoding a gene product of interest. By contrast, a regulated genetic circuit may contain a nucleic acid with an inducible promoter that activates gene expression in response to specific stimuli or that inactivates gene expression in response to specific stimuli.

Genetic Elements

Genetic circuits of the present disclosure contain at least one genetic element that can regulate gene/protein expression. A “genetic element,” as used herein, refers to a nucleotide sequence that has a role in gene expression. For example, nucleic acids (e.g., recombinant nucleic acids) encoding proteins, promoters, enhancers and terminators are considered to be genetic elements.

Nucleic Acids

As used herein, the term “nucleic acid” refers to at least two nucleotides covalently linked together, and in some instances, may contain phosphodiester bonds (e.g., a phosphodiester “backbone”). In some embodiments, a nucleic acid of the present disclosure may be considered to be a nucleic acid analog, which may contain other backbones comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages, and/or peptide nucleic acids. Nucleic acids of the present disclosure may be naturally occurring, recombinant or synthetic. “Recombinant nucleic acids” may refer to molecules that are constructed by joining nucleic acid molecules and, in some embodiments, can replicate in a living cell. “Synthetic nucleic acids” may refer to molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.

The nucleic acids may be single-stranded (ss) or double-stranded (ds), as specified, or may contain portions of both single-stranded and double-stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, and isoguanine.

Promoters

The genetic circuits of the present disclosure may contain nucleic acids with promoter sequences, or promoters, operably linked to a nucleotide sequence encoding a gene product of interest. As used herein, a “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain subregions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof.

A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.

A promoter may be classified as strong or weak according to its affinity for RNA polymerase (and/or sigma factor); this is related to how closely the promoter sequence resembles the ideal consensus sequence for the polymerase. The strength of a promoter may depend on whether initiation of transcription occurs at that promoter with high or low frequency. Different promoters with different strengths may be used to construct genetic circuits with different levels of gene/protein expression (e.g., the level of expression initiated from a weak promoter is lower than the level of expression initiated from a strong promoter).

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon of a given gene or sequence. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.

In some embodiments, a coding nucleic acid segment may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic cell; and synthetic promoters or enhancers that are not “naturally occurring” such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see U.S. Pat. Nos. 4,683,202 and 5,928,906).

Inducible Promoters

As used herein, an “inducible promoter” is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by or contacted by an inducer or inducing agent. An “inducer” or “inducing agent” may be endogenous or a normally exogenous condition, compound or protein that contacts a genetic circuit in such a way as to be active in inducing transcriptional activity from the inducible promoter.

Inducible promoters for use in accordance with the present disclosure function in a bacterial cell. Examples of inducible promoters for use herein include, without limitation, bacteriophage promoters (e.g. Pls1con, T3, T7, SP6) and bacterial promoters (e.g. Pbad, PmgrB, PL1acO, Ptrc2, PLtetO, Plac/ara, Ptac, Pm). Examples of bacterial promoters for use in accordance with the present disclosure include, without limitation, positively regulated E. coli promoters such as positively regulated σ⁷⁰ promoters (e.g., inducible pBad/araC promoter, Lux cassette right promoter, modified lamdba Prm promote, plac Or2-62 (positive), pBad/AraC with extra REN sites, pBad, P(Las) TetO, P(Las) CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), as promoters (e.g., Pdps), σ³² promoters (e.g., heat shock) and σ⁵⁴ promoters (e.g., glnAp2); negatively regulated E. coli promoters such as negatively regulated σ⁷⁰ promoters (e.g., Promoter (PRM+), modified lamdba Prm promoter, TetR-TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ, RecA_DlexO_DLacO1, dapAp, FecA, Pspac-hy, pcI, plux-cI, plux-lac, CinR, CinL, glucose controlled, modifed Pr, modifed Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS), EmrR_regulated, BetI_regulated, pLac_lux, pTet_Lac, pLac/Mnt, pTet/Mnt, LsrA/cI, pLux/cI, LacI, LacIQ, pLacIQ1, pLas/cI, pLas/Lux, pLux/Las, pRecA with LexA binding site, reverse BBa_R0011, pLacI/ara-1, pLacIq, rrnB P1, cadC, hns, PfhuA, pBad/araC, nhaA, OmpF, RcnR), σ^(S) promoters (e.g., Lutz-Bujard LacO with alternative sigma factor σ³⁸), σ³² promoters (e.g., Lutz-Bujard LacO with alternative sigma factor σ³²), and σ⁵⁴ promoters (e.g., glnAp2); negatively regulated B. subtilis promoters such as repressible B. subtilis σ ^(A) promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and σ^(B) promoters. Other inducible bacterial promoters may be used in accordance with the present disclosure.

The administration or removal of an inducer results in a switch between activation and inactivation of the transcription of the operably linked nucleic acid sequence (e.g., nucleic acid encoding a gene product of interest). Thus, as used herein, the active state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is actively driving transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is expressed). Conversely, the inactive state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is not actively driving transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is not expressed).

An inducible promoter for use in accordance with the present disclosure may be induced by (or repressed by) one or more physiological condition(s), such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agent(s). The extrinsic inducer or inducing agent may comprise, without limitation, amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or combinations thereof.

Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

In some embodiments, the inducer used in accordance with the present disclosure is an N-acyl homoserine lactone (AHL), which is a class of signaling molecules involved in bacterial quorum sensing. Quorum sensing is a method of communication between bacteria that enables the coordination of group based behavior based on population density. AHL can diffuse across cell membranes and is stable in growth media over a range of pH values. AHL can bind to transcriptional activators such as LuxR and stimulate transcription from cognate promoters. In some embodiments, the inducer used in accordance with the present disclosure is anhydrotetracycline (aTc), which is a derivative of tetracycline that exhibits no antibiotic activity and is designed for use with tetracycline-controlled gene expression systems, for example, in bacteria.

Other inducible promoter systems are known in the art and may be used in accordance with the present disclosure.

Enhancers

In some embodiments of the present disclosure, a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence downstream of the promoter. The enhancer may be located at any functional location before or after the promoter.

Terminators

In some embodiments, a genetic circuit may contain a terminator sequence, or terminator. A “terminator,” as used herein, is a nucleic acid sequence that causes transcription to stop. A terminator may be unidirectional or bidirectional. It is comprised of a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase. A terminator sequence prevents transcriptional activation of downstream nucleic acid sequences by upstream promoters. Thus, in certain embodiments, a terminator that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable gene/protein expression levels.

The most commonly used type of terminator is a forward terminator. When placed downstream of a nucleic acid sequence that is usually transcribed, a forward transcriptional terminator will cause transcription to abort. In some embodiments, bidirectional transcriptional terminators are provided, which usually cause transcription to terminate on both the forward and reverse strand. In some embodiments, reverse transcriptional terminators are provided, which usually terminate transcription on the reverse strand only.

In prokaryotic systems, terminators usually fall into two categories (1) rho-independent terminators and (2) rho-dependent terminators. Rho-independent terminators are generally composed of palindromic sequence that forms a stem loop rich in G-C base pairs followed by several T bases. Without wishing to be bound by theory, the conventional model of transcriptional termination is that the stem loop causes RNA polymerase to pause, and transcription of the poly-A tail causes the RNA:DNA duplex to unwind and dissociate from RNA polymerase.

Terminators for use in accordance with the present disclosure include any terminator of transcription described herein or known to one of ordinary skill in the art. Examples of terminators include, without limitation, the termination sequences of genes such as, for example, the bovine growth hormone terminator, and viral termination sequences such as, for example, the T0 terminator, the TE terminator, Lambda T1 and the T1T2 terminator found in bacterial systems. In some embodiments, the termination signal may be a sequence that cannot be transcribed or translated, such as those resulting from a sequence truncation.

Other genetic elements are known in the art and may be used in accordance with the present disclosure.

Recombinase-Based Genetic Circuits

In some embodiments of the present disclosure, the endogenous bacterial cells are functionalized with recombinase-based genetic circuits. A “recombinase,” as used herein, is a site-specific enzyme that recognizes short DNA sequence(s), which sequence(s) are typically between about 30 base pairs (bp) and 40 bp, and that mediates the recombination between these recombinase recognition sequences, which results in the excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences. For example, in some embodiments, recombinant bacteriophages and/or phagemids of the present disclosure may be engineered to deliver at least two genetic circuits, one containing a nucleic acid with an inducible promoter operably linked to a nucleic acid encoding a recombinase, and the other containing a nucleic acid with a promoter operably linked to a nucleotide sequence encoding a gene product of interest and optionally containing a terminator, wherein at least one of the promoter and terminator is flanked by a forward and a reverse recognition site of the recombinase. In such embodiments, expression of the gene product of interest of one circuit is regulated by recombinase activity, or inactivity, of the other circuit.

Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases), based on distinct biochemical properties. Serine recombinases and tyrosine recombinases are further divided into bidirectional recombinases and unidirectional recombinases. Examples of bidirectional serine recombinases for use herein include, without limitation, β-six, CinH, ParA and γδ; and examples of unidirectional serine recombinases include, without limitation, Bxb1, ϕC31, TP901, TG1, φBT1, R4, φRV1, φFC1, MR11, A118, U153 and gp29. Examples of bidirectional tyrosine recombinases for use herein include, without limitation, Cre, FLP, and R; and unidirectional tyrosine recombinases include, without limitation, Lambda, HK101, HK022 and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange.

The outcome of recombination depends, in part, on the location and orientation of two short repeated DNA sequences that are to be recombined, typically less than 30 bp long. Recombinases bind to these repeated sequences, which are specific to each recombinase, and are herein referred to as “recombinase recognition sequences” or “recombinase recognition sites.” Thus, as used herein, a recombinase is “specific for” a recombinase recognition site when the recombinase can mediate inversion or excision between the repeat nucleotide sequences. As used herein, a recombinase may also be said to recognize its “cognate recombinase recognition sites,” which flank an intervening genetic element (e.g., promoter, terminator, or output nucleic acid sequence). A genetic element is said to be “flanked” by recombinase recognition sites when the element is located between and immediately adjacent to two repeated nucleotide sequences.

Recombinases can also be classified as irreversible or reversible. As used herein, an “irreversible recombinase” (also referred to as a “unidirectional recombinase”) refers to a recombinase that can catalyze recombination between two complementary recombination sites, but cannot catalyze recombination between the hybrid sites that are formed by this recombination without the assistance of an additional factor. Thus, an “irreversible recognition site” refers to a recombinase recognition site that can serve as the first of two nucleotide recognition sequences for an irreversible recombinase and that is modified to a hybrid recognition site following recombination at that site. A “complementary irreversible recognition site” refers to a recombinase recognition site that can serve as the second of two nucleotide recognition sequences for an irreversible recombinase and that is modified to a hybrid recombination site following homologous recombination at that site.

Irreversible recombinases, and nucleic acids that encode the irreversible recombinases, are described in the art and can be obtained using routine methods. Examples of irreversible recombinases include, without limitation, phiC31 ((φC31) recombinase, coliphage P4 recombinase (Ow & Ausubel, J. Bacteriol. 155, 704-713 (1983)), coliphage lambda integrase (Lorbach et al., J. Mol. Biol., 296, 1175-81 (2000)), Listeria A118 phage recombinase (Loessner et al., Mol. Micro. 35, 324-340 (2000)), and actinophage R4 Sre recombinase (Matsuura et al., J Bacteriol. 178, 3374-3376 (1996)), HK101, HK022, pSAM2, Bxb1, TP901, TG1, φBT1, φRV1, φFC1, MR11, U153 and gp29.

Conversely, a “reversible recombinase” refers to a recombinase that can catalyze recombination between two complementary recombinase recognition sites and, without the assistance of an additional factor, can catalyze recombination between the sites that are formed by the initial recombination event, thereby reversing it. The product-sites generated by recombination are themselves substrates for subsequent recombination. Examples of reversible recombinase systems include, without limitation, the Cre-lox and the Flp-frt systems, R, β-six, CinH, ParA and γδ.

In some embodiments, the recombinase is serine recombinase. Thus, in some embodiments, the recombinase is considered to be irreversible. In some embodiments, the recombinase is a tyrosine recombinase. Thus, in some embodiments, the recombinase is considered to be reversible.

The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the present disclosure. The complexity of the genetic circuits of the present disclosure can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities (Groth, A. C. & Calos, M. P. J Mol Biol 335, 667-678, (2004); Gordley, R. M., et al. Proc Natl Acad Sci USA 106, 5053-5058 (2009)). Other examples of recombinases that are useful in the genetic circuits described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the present disclosure.

Gene Products

The present disclosure contemplates recombinant bacteriophages and/or phagemids delivery of genetic circuits that encode one or more of a variety of gene products. As used herein, a “gene product” may refer to a protein product or RNA product that may be used, for example, as a therapeutic molecule or as a diagnostic or reporter molecule. Representative gene products for genetic circuits of the present disclosure include, without limitation, therapeutic proteins, reporter proteins, transcriptional repressors, transcriptional activators, enzymes, receptor proteins, ligand proteins, RNAs, riboswitches, RNA interference (e.g., shRNA, siRNA, microRNA) molecules and recombinases.

Therapeutic Molecules

Methods of the present disclosure, in some embodiments, may comprise delivering to endogenous bacterial cells a recombinant bacteriophage and/or phagemid that is engineered to contain at least one genetic circuit that expresses a therapeutic molecule. Therapeutic molecules include therapeutic proteins. Therapeutic molecules of the present disclosure may be used to, for example, replace a protein that is deficient or abnormal, augment an existing biological pathway, provide a novel function or activity, interfere with a molecule or organism, and/or deliver other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic molecule contemplated by the present disclosure include, without limitation, antibodies, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Other examples include those that bind non-covalently to target (e.g., monoclonal antibodies), those that affect covalent bonds (e.g., enzymes), and those that exert activity without specific interactions (e.g., serum albumin).

Also contemplated herein are therapeutic molecules, e.g., recombinant therapeutic proteins, used to treat, for example, cancers, immune disorders, infections and/or other diseases. Engineered proteins, including bispecific mAbs and multispecific fusion proteins, and proteins with optimized pharmacokinetics are also contemplated by the present disclosure.

In some embodiments, the therapeutic proteins is Etanercept, Bevacizumab, Rituximab, Adalimumab, Infliximab, Trastuzumab, Insulin glargine, Epoetin alfa, Pegfilgrastim, Ranibizumab, Darbepoetin alfa, Interferon beta-1a, Interferon beta-1a. Insulin aspart, Rhu insulin, Octocog alfa, Insulin lispro, Cetuximab, Peginterferon alfa-2a, Interferon beta-1b, Eptacog alfa, Insulin aspart, OnabotulinumtoxinA, Epoetin beta, Rec antihemophilic factor, Filgrastin, Insulin detemir, Natalizumab, Insulin (humulin) or Palivizumab.

Examples of antibodies, antibody fragments, and/or Fc fusion proteins that may be encoded by the genetic circuits of the present disclosure include, without limitation, Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (or tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, Cedelizumab, Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Concizumab, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN, Ticilimumab (or tremelimumab), Tildrakizumab, Tigatuzumab, TNX-, Tocilizumab (or atlizumab), Toralizumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, TRBS, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vantictumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab and Zolimomab aritox.

Other examples of Fc fusion proteins that may be encoded by the genetic circuits of the present disclosure include, without limitation, Etanercept, Alefacept, Abatacept, Rilonacept, Romiplostim, Belatacept and Aflibercept.

Examples of anticoagulants and/or blood factors that may be encoded by the genetic circuits of the present disclosure include, without limitation, Protein C, Protein S, and antithrombin, Factors I-VIII, prothrombinase, prothrombin, thrombin von Willebrand Factor (vWF), fibrinogen, fibrin and fibrinopeptides.

Examples of bone morphogenetic proteins (BMPs) that may be encoded by the genetic circuits of the present disclosure include, without limitation, BMP1-BMP7, BMP8a, BMP8b, BMP10, and BMP15.

Examples of enzymes that may be encoded by the genetic circuits of the present disclosure include, without limitation, any of the enzymes assigned an Enzyme Commission Number (EC) number (e.g., EC1-EC6) by the International Union of Biochemistry and Molecular Biology (IUBMB) (Webb, Edwin C. Enzyme nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes.

San Diego: Published for the International Union of Biochemistry and Molecular Biology by Academic Press. ISBN 0-12-227164-5 (1992), incorporated by reference herein). Other examples include: styrene monooxygenase (StyAB), toluene dioxygenase (TODC1C2AB), luciferase and lactase. In some embodiments, the enzyme is toluene dioxygenase. In some embodiments, the enzyme is styrene monoxygenase.

Examples of growth factors that may be encoded by the genetic circuits of the present disclosure include, without limitation, Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-α), Transforming growth factor beta(TGF-0), Tumor necrosis factor-alpha(TNF-α), Vascular endothelial growth factor (VEGF), placental growth factor (PlGF), Foetal Bovine Somatotrophin (FBS) and IL-1-IL7.

Examples of peptide hormones that may be encoded by the genetic circuits of the present disclosure include, without limitation, Amylin (or Islet Amyloid Polypeptide), Antimullerian hormone (or Müllerian inhibiting factor or hormone), Adiponectin, Adrenocorticotropic hormone (or corticotropin), Angiotensinogen and angiotensin, Antidiuretic hormone (or vasopressin, arginine vasopressin), Atrial-natriuretic peptide (or atriopeptin), Brain natriuretic peptide, Calcitonin, Cholecystokinin, Corticotropin-releasing hormone, Enkephalin, Endothelin, Erythropoietin, Follicle-stimulating hormone, Galanin, Gastrin, Ghrelin, Glucagon, Gonadotropin-releasing hormone, Growth hormone-releasing hormone, Human chorionic gonadotropin, Human placental lactogen, Growth hormone, Inhibin, Insulin, Insulin-like growth factor (or somatomedin), Leptin, Lipotropin, Luteinizing hormone, Melanocyte stimulating hormone, Motilin, Orexin, Oxytocin, Pancreatic polypeptide, Parathyroid hormone, Prolactin, Prolactin releasing hormone, Relaxin, Renin, Secretin, Somatostatin, Thrombopoietin, Thyroid-stimulating hormone (or thyrotropin), and Thyrotropin-releasing hormone.

Examples of interferons (IFNs) that may be encoded by the genetic circuits of the present disclosure include, without limitation, IFN-α, IFN-β, IFN-ω and IFN-γ.

Examples of interleukins that may be encoded by the genetic circuits of the present disclosure include, without limitation, interleukin 1-17. In some embodiments, the interleukin is Interleukin-4, Interleukin-6, Interleukin-10, Interleukin-11 or Interleukin-13.

Other examples of therapeutic proteins that may be encoded by the genetic circuits of the inventionpresent disclosure include, without limitation, Insulin (blood glucose regulator), Pramlintide acetate (glucose control), Growth hormone GH (growth failure), Pegvisoman (growth hormone receptor antagonist), Mecasermin (IGF1, growth failure), Factor VIII (coagulation factor), Factor IX (coagulation factor, Protein C concentrate (anti-coagulation), al-proteinase inhibitor (anti-trypsin inhibitor), Erythropoietin (stimulates erythropoiesis), Filgrastim (granulocyte colony-stimulating factor, G-CSF; stimulates neutrophil proliferation), Sargramostim36, 37 (granulocytemacrophage colony-stimulating factor, GM-CSF), Oprelvekin (interleukin11, IL11), Human follicle-stimulating hormone (FSH), Human chorionic gonadotropin (HCG), Lutropin-α (human luteinizing hormone), Interleukin 2 (IL2), Interleukin-1 Receptor Agonist, Denileukin diftitox (fusion of IL2 and Diphtheria toxin), Interferon alfacon 1 (consensus interferon), Interferon-α2a (IFNα2a), Interferon-α2b (IFNα2b), Interferon-αn3 (IFNαn3), Interferon-β1α (rIFN-β), Interferon-β1b (rIFN-β), Interferon-γ1b (IFNγ), Salmon calcitonin (32-amino acid linear polypeptide hormone), Teriparatide (part of human parathyroid hormone 1-34 residues), Exenatide (Incretin mimetic with actions similar to glucagon-like peptide 1), Octreotide (octapeptide that mimics natural somatostatin), Dibotermin-α (recombinant human bone morphogenic protein 2), Recombinant human bone morphogenic protein 7, Histrelin acetate (gonadotropin-releasing hormone; GnRH), Palifermin (Keratinocyte growth factor, KGF), Becaplermin (platelet-derived growth factor, PDGF), Nesiritide (recombinant human B-type natriuretic peptide), Lepirudin (recombinant variant of hirudin, another variant is Bivalirudin), Anakinra (interleukin 1 (IL1) receptor antagonist), Enfuviritide (an HIV-1 gp41-derived peptide), β-Glucocerebrosidase (hydrolyzes to glucose and ceramide), Alglucosidase-α (degrades glycogen), Laronidase (digests glycosaminoglycans within lysosomes), Idursulfase (cleaves O-sulfate preventing GAGs accumulation), Galsulfase (cleave terminal sulphage from GAGs), Agalsidase-β (human α-galactosidase A, hydrolyzes glycosphingolipids), Lactase (digest lactose), Pancreatic enzymes (lipase, amylase, protease; digest food), Adenosine deaminase (metabolizes adenosine), Tissue plasminogen activator (tPA, serine protease involved in the breakdown of blood clots), Factor VIIa (serine protease, causes blood to clot), Drotrecogin-α (serine protease, human activated protein C), Trypsin (serine protease, hydrolyzes proteins), Botulinum toxin type A (protease, inactivates SNAP-25 which is involved in synaptic vesicle fusion), Botulinum toxin type B (protease that inactivates SNAP-25 which is involved in synaptic vesicle fusion), Collagenase (endopeptidase, digest native collagen), Human deoxyribonuclease I (endonuclease, DNase I, cleaves DNA), Hyaluronidase (hydrolyzes hyaluronan), Papain (cysteine protease, hydrolyzes proteins), L-Asparaginase (catalyzes the conversion of L-asparagine to aspartic acid and ammonia), Rasburicase (urate oxidase, catalyzes the conversion of uric acid to allantoin), Streptokinase (Anistreplase is anisoylated plasminogen streptokinase activator complex (APSAC)), and Antithrombin III (serine protease inhibitor).

Reporter Molecules

In some embodiments, the genetic circuits of the present disclosure may encode a “reporter.” As used herein, a reporter refers to a protein that can be used to measure gene expression and generally produce a measurable signal such as fluorescence, luminescence or color. The presence of a reporter in a cell or organism is readily observed. For example, fluorescent proteins (e.g., green fluorescent protein (GFP)) cause a cell to fluoresce when excited with light of a particular wavelength, luciferases cause a cell to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate to a colored product. In some embodiments, reporters may be used to quantify the strength or activity of the input received by the systems of the present disclosure. In some embodiments, reporters can be fused in-frame to other protein coding sequences to identify where a protein is located in a cell or organism. Reporters for use in accordance with the present disclosure include any reporter described herein or known to one of ordinary skill in the art.

There are several different ways to measure or quantify a reporter depending on the particular reporter and what kind of characterization data is desired. In some embodiments, microscopy can be a useful technique for obtaining both spatial and temporal information on reporter activity, particularly at the single cell level. In some embodiments, flow cytometers can be used for measuring the distribution in reporter activity across a large population of cells. In some embodiments, plate readers may be used for taking population average measurements of many different samples over time. In some embodiments, instruments that combine such various functions, may be used, such as multiplex plate readers designed for flow cytometers, and combination microscopy and flow cytometric instruments.

Fluorescent proteins may be used for visualizing or quantifying gene product expression. Fluorescence can be readily quantified using a microscope, plate reader or flow cytometer equipped to excite the fluorescent protein with the appropriate wavelength of light. Several different fluorescent proteins are available, thus multiple gene expression measurements can be made in parallel. Examples of genes encoding fluorescent proteins that may be used in accordance with the present disclosure include, without limitation, those proteins provided in U.S. Patent Application No. 2012/0003630 (see Table 59), incorporated herein by reference.

Luciferases may also be used for visualizing or quantifying gene product expression, particularly for measuring low levels of gene expression, as cells tend to have little to no background luminescence in the absence of a luciferase. Luminescence can be readily quantified using a plate reader or luminescence counter. Examples of genes encoding luciferases for that may be used in accordance with the present disclosure include, without limitation, dmMyD88-linker-Rluc, dmMyD88-linker-Rluc-linker-PEST191, luxAB, NanoLuc, Renilla remformis luciferase, and firefly luciferase (from Photinus pyralis).

Enzymes that produce colored substrates (“colorimetric enzymes”) may also be used for visualizing or quantifying gene product expression. Enzymatic products may be quantified using spectrophotometers or other instruments that can take absorbance measurements including plate readers. Like luciferases, enzymes such as β-galactosidase can be used for measuring low levels of gene expression because they tend to amplify low signals. Examples of genes encoding colorimetric enzymes that may be used in accordance with the present disclosure include, without limitation, lacZ alpha fragment, lacZ (encoding beta-galactosidase, full-length), and xylE.

Transcriptional Activators and/or Repressors

In some embodiments, the genetic circuits of the present disclosure may encode a transcriptional activator or repressor, the production of which can result in a further change in state of the cell, and provide additional input signals to subsequent or additional genetic circuits. Transcriptional regulators either activate or repress transcription from cognate promoters. Transcriptional activators typically bind nearby to transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and sterically hinder transcriptional initiation by RNA polymerase. Other transcriptional regulators serve as either an activator or a repressor depending on where it binds and cellular conditions. Transcriptional regulators for use in accordance with the present disclosure include any transcriptional regulator described herein or known to one of ordinary skill in the art. Examples of genes encoding transcriptional regulators that may be used in accordance with the present disclosure include, without limitation, those regulators provided in U.S. Patent Application No. 2012/0003630 (see Table 63), incorporated herein by reference.

Enzymes

In some embodiments, the genetic circuits of the present disclosure may encode an enzyme. In some embodiments, an enzyme is used as a response to a particular input. For example, in response to a particular input received by a genetic circuit of the present disclosure, such as a certain range of toxin concentration present in the environment, the system may activate transcription of nucleic acid sequence that encodes an enzyme that can degrade or otherwise destroy the toxin. In some embodiments, enzymes may be “biosynthetic enzymes” that catalyze the conversion of substrates to products. For example, such biosynthetic enzymes can be used in accordance with the present disclosure to assemble pathways that produce or degrade useful chemicals and materials, in response to specific signals. These combinations of enzymes can reconstitute either natural or synthetic biosynthetic pathways. Enzymes for use in accordance with the present disclosure include any enzyme described herein or known to one of ordinary skill in the art. Examples of genes encoding enzymes that may be used in accordance with the present disclosure include, without limitation, those provided in U.S. Patent Application No. 2012/0003630, incorporated herein by reference.

Receptors, Ligands, and Lytic Proteins

In some embodiments, the genetic circuits of the present disclosure may encode a receptor, ligand or lytic protein. Receptors tend to have three domains: an extracellular domain for binding ligands such as proteins, peptides or small molecules, a transmembrane domain and an intracellular or cytoplasmic domain, which frequently can participate in some sort of signal transduction event such as phosphorylation. In some embodiments, transporters, channels or pumps are used as output products. Transporters are membrane proteins responsible for transport of substances across the cell membrane. Channels are made up of proteins that form transmembrane pores through which selected ions can diffuse. Pumps are membrane proteins that can move substances against their gradients in an energy-dependent process known as active transport. In some embodiments, nucleic acid sequences encoding proteins and protein domains whose primary purpose is to bind other proteins, ions, small molecules, and other ligands may be used in accordance with the present disclosure. Receptors, ligands and lytic proteins for use in accordance with the present disclosure include any receptor, ligand and lytic protein, described herein or known to one of ordinary skill in the art. Examples of genes encoding receptors, ligands and lytic proteins that may be used in accordance with the present disclosure include, without limitation, those provided in U.S. Patent Application No. 2012/0003630 (see Table 73), incorporated herein by reference.

Antimicrobial Proteins/Peptides

In some embodiments, the at least one genetic circuit may express an antimicrobial protein and/or peptide. However, it is to be understood that such antimicrobial proteins and/or peptides may be specifically excluded from the genetic circuits of the present disclosure. Thus, in some embodiments, the at least one genetic circuit does not express an antimicrobial protein or peptide. Examples of antimicrobial proteins and/or peptides contemplated herein include gene modules that encode instructions for cell death, or bactericidal proteins. Examples of such gene modules include pemI-pemK genes of plasmid R100, the phd-doc genes of phage P1, the ccdA-ccdB genes of plasmid F, mazE-mazF (or chpAI-chpAK), sof-gef, kicA-kicB, relB-relE, chpBI-chpBK and gef. Other examples of antimicrobial proteins and/or peptides include, without limitation, bacteriocins, hydramacin-1, cecropins, moricins, papiliocins, poneratoxins, mastoparans, melittins, spinigerins, cupiennins, oxyopinins, magainins, dermaseptins, cathelicidins, defensins and protegrins. Other antimicrobial proteins and/or peptides are also contemplated by the present disclosure.

Engineering Genetic Circuits

The genetic circuits of the present disclosure may be engineered using, for example, standard molecular cloning methods (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. M., et al., New York: John Wiley & Sons, 2006; Gibson, D. G., et al., Nature Methods 6(5):343-345 (2009), the teachings of which relating to molecular cloning are herein incorporated by reference).

Uses of Functionalized Bacteria

A genetic circuit, in accordance with the present disclosure, may be engineered to impart to endogenous bacteria a variety of functions such as, for example, the ability to express therapeutic molecules, to modify cellular functions, to create cellular responses to environmental conditions, and/or influence cellular development. By providing a method of delivering rational, controllable genetic circuits to bacterial cells of a microbiome in vivo, in some embodiments, the present disclosure permits the use of such cells as engineered systems to perform a vast range of useful functions that may greatly benefit the host organism.

The functionalized endogenous bacterial cells of the present disclosure may be used for a variety of applications, including, without limitation, bioremediation, biosensing and biomedical therapeutics. In some embodiments, the genetic circuits may be used to build in the endogenous bacterial cells multiplexed cellular switches for gene expression or synthetic differentiation cascades. In some embodiments, the genetic circuits may be used to regulate (e.g., activate and/or deactivate) in the same cell, at the same time or sequentially, transcription of various molecules of interest.

In some embodiments, the methods of the present disclosure may be used to deliver molecules that treat, or alleviate the symptoms associated with, a condition such as, for example, cancer (e.g., gastrointestinal cancer), immune disorders, infections and/or other diseases (e.g., gastrointestinal disease). Thus, in some embodiments, the present disclosure provides methods of delivering to a subject a recombinant bacteriophage (e.g., coliphage) and/or phagemid engineered to contain at least one genetic circuit. In some embodiments, the genetic circuit comprises a nucleic acid with a promoter operably linked to a nucleotide sequence encoding a therapeutic molecule. The therapeutic molecule may be, for example, an antibody, antibody-based drug, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin or thrombolytic.

Examples of gastrointestinal cancers include, without limitation, cancers of the esophagus, gallbladder, liver, pancreas, stomach, small intestine, large intestine (colon) and rectum.

Examples of immune diseases include, without limitation, Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal & neuronal neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic fatigue syndrome**, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Type 1 diabetes, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo and Wegener's granulomatosis (also referred to as Granulomatosis with Polyangiitis (GPA)).

The methods of the present disclosure may be used to deliver molecules that treat, or alleviate the symptoms associated with, gastrointestinal diseases. Examples of gastrointestinal diseases include, without limitation, Crohn's disease, ulcerative colitis and colon cancer.

Crohn's disease is a condition of chronic inflammation potentially involving any location of the gastrointestinal tract, but it frequently affects the end of the small bowel and the beginning of the large bowel. In Crohn's disease, all layers of the intestine may be involved, and there can be normal healthy bowel in between patches of diseased bowel. Symptoms include persistent diarrhea (loose, watery, or frequent bowel movements), cramping abdominal pain, fever, and, at times, rectal bleeding. Loss of appetite and weight loss also may occur. However, the disease is not always limited to the gastrointestinal tract; it can also affect the joints, eyes, skin and liver. Fatigue is another common symptom. In some embodiments, the methods of the present disclosure are used to deliver to endogenous bacterial cells a genetic circuit that expresses gene(s) encoding the monoclonal antibody, infliximab, Interleukin-4, Interleukin-6, Interleukin-10, Interleukin-11, Interleukin-13, or Interleukin-1 Receptor Antagonist. In some embodiments, the present disclosure provides methods of delivering to a subject having Crohn's disease a recombinant bacteriophage (e.g., coliphage) and/or phagemid engineered to contain at least one genetic circuit. In some embodiments, the genetic circuit comprises a nucleic acid with a promoter operably linked to a nucleotide sequence encoding a monoclonal antibody (e.g., infliximab), Interleukin-4, Interleukin-6, Interleukin-10, Interleukin-11, Interleukin-13, or Interleukin-1 Receptor Antagonist.

Ulcerative colitis is a chronic gastrointestinal disorder that is limited to the large bowel (the colon). Ulcerative colitis does not affect all layers of the bowel, but only affects the top layers of the colon in an even and continuous distribution. The first symptom of ulcerative colitis is a progressive loosening of the stool. The stool is generally bloody and may be associated with cramping abdominal pain and severe urgency to have a bowel movement. The diarrhea may begin slowly or quite suddenly. Loss of appetite and subsequent weight loss are common, as is fatigue. In cases of severe bleeding, anemia may also occur. In addition, there may be skin lesions, joint pain, eye inflammation and liver disorders. Children with ulcerative colitis may fail to develop or grow properly. In some embodiments, the methods of the present disclosure are used to deliver to endogenous bacterial cells a genetic circuit that expresses gene(s) encoding Interleukin-4, Interleukin-6, Interleukin-10, Interleukin-11, Interleukin-13, or Interleukin-1 Receptor Antagonist. In some embodiments, the present disclosure provides methods of delivering to a subject having ulcerative colitis a recombinant bacteriophage (e.g., coliphage) and/or phagemid engineered to contain at least one genetic circuit. In some embodiments, the genetic circuit comprises a nucleic acid with a promoter operably linked to a nucleotide sequence encoding a monoclonal antibody (e.g., infliximab), Interleukin-4, Interleukin-6, Interleukin-10, Interleukin-11, Interleukin-13, or Interleukin-1 Receptor Antagonist.

In some embodiments, the present disclosure provides methods of delivering to a subject having colon cancer a recombinant bacteriophage (e.g., coliphage) and/or phagemid that is engineered to contain at least one genetic circuit.

EXAMPLES Example 1. Indigo Networks on Plasmids

Two different oxidative enzymes were chosen for the purpose of converting indole, a metabolite of Tryptophan, to indigo, an insoluble dye. The first enzyme, toluene dioxygenase, is found in the organism Pseudomonas putida and is responsible for catalyzing the oxygenation of various ethenes, butenes, and propenes (Woo et al. Journal of Microbiological Methods 40, 181-191 (2000)). Toulene dioxygenase (e.g., TODC1C2AB) is a multicomponent enzyme that primarily converts indole to indolediol, which is then dimerized spontaneously to indigo. Styrene monooxygenase is also found in Pseudomonas putida and is responsible for degrading various styrene molecules (O'connor et al. Applied and Environmental Microbiology 63, 4287-4291 (1997)). Styrene monooxygenase is made up of two components, StyA and StyB. StyA slowly catalyzes the conversion of indole to indole oxide, which can naturally form 2-oxindole before dimerizing to indigo. StyB converts indole oxide to indoxyl, which forms indigo at a much faster pace. Both enzymes are capable of oxidizing indole and subsequently catalyzing its dimerization to form indigo in vivo. Once formed, the indigo can diffuse through the cell membrane and into the extracellular fluid, where at high enough concentrations it aggregates (Pinero-Fernandez et al. Journal of Bacteriology 193:1793-1798 (2011)).

The indole networks were designed to be either constitutive or inducible. The PltetO promoter is constitutive unless inhibited by the tetR protein, which is not expressed in wild type E. coli. The Pbad promoter is induced by the small molecule arabinose in a dose-dependent manner. Ribosomal binding sites (RBSs) were optimized for high expression by using a RBS calculator.

Cloning

Cloning was accomplished through standard methods. The oxidative enzymes were obtained by PCR amplification from published strains, using primers specific for the 5′ and 3′ ends. The 5′ primer included the designed RBS. Promoter and terminator elements were obtained by PCR from published library plasmids described by Litcofsky et al. (Nat Meth 9(11):1077-1080 (2012). These purified components were then digested and ligated into the multiple cloning site of the pKE2-MCS cloning plasmid also described by Litcofsky et al. The ligated plasmid was then transformed into the test strain MgPro, which is designed to overexpress the tetR and lacI cassettes.

Indigo Production

Indigo networks were grown overnight in lysogeny broth (LB), then diluted 1:1000 in M9 minimal media (e.g., M9 salts, MgSO₄, CaCl₂), carbon source such as, e.g., glycerol, or glucose). At OD ˜0.2-0.3, 0.5 μM indole and the respective inducer were added to the media, and samples were collected every hour for four hours. The samples were spun down for one minute at 13 k rpm, aspirated and brought up in dimethyl sulfoxide (DMSO). After vortexing, the samples were spun again at 13 k rpm before being read on a plate reader at 610 nm. Pbad was induced with 0.01% arabinose. PltetO was induced with 0.5 μg/mL aTe.

Results

As shown in FIGS. 1A and 1B, indigo was produced using both the constitutive PLtet0 and the inducible Pbad promoter systems for the production of toluene dioxygenase, TODC1C2AB. Similarly, as shown in FIGS. 2A and 2B, indigo was produced using both the constitutive PLtet0 and the inducible Pbad promoter systems for the production of styrene monooxygenase, StyAB. By contrast, as shown in FIG. 2C, little indigo was produced using both the constitutive PLtet0 and the inducible Pbad promoter systems for the production of StyA.

Example 2. Transducible Expression of styAB Using Engineered M13 Phage

One important aspect of engineering genetic circuits that has yet to be thoroughly explored is the ability to deliver and introduce functioning circuits into cells under in situ or in vivo conditions. Embodiments of the present disclosure provide engineered M13 bacteriophage for delivery of such circuits into a target population. The recombinant bacteriophages provided herein are used to deliver genetic circuits in vivo by targeting bacteria in the intestines of mice. This technology has broad applications as a platform for circuit delivery in order to apply some of the exciting creations of synthetic biology such as intricate biosensors to real-life animal models.

M13mpl8 replicative form DNA was acquired from NEB (#N4018S) and used for the genetic manipulations of M13 phage. E. coli XLI 0, a cloning strain of bacteria harboring the F plasmid required for M13 infection, was used for cloning and M13 propagation and was grown in LB with 25 μg/mL chloramphenicol. Briefly, the desired genetic circuits were amplified from template plasmid and cloned in vitro into the multiple cloning site of M13mpl8. The ligation products were transformed and plated in 0.7% LB top agar along with 200 μL of additional bacteria from overnight culture to generate a bacterial lawn with plaques corresponding to M13 infection foci. Plaques were picked into a 1:3 dilution of overnight XL10 culture in fresh LB and grown for 4 hours to overnight at 37° C. Culture supernatants were sterile-filtered to collect crude phage preparations, and pellets were subjected to alkaline lysis using QIAGEN® Miniprep Kits for sequencing.

For initial phenotype verification, an overnight culture of E. coli EMG2 (F+) was washed once and subsequently resuspended in M9 plus glucose. Cultures of E. coli MG1655Pro harboring the template plasmids used for cloning into M13 were prepared as positive controls in the same manner. Washed cell suspensions were diluted four-fold into fresh M9 plus glucose with 10 mM arabinose (ara), 250 ng/μL anhydrotetracycline (aTc), or neither. Phage supernatants as prepared above were added at 1:100, and cultures were incubated at 37° C. with shaking. After 4 hours, indole was supplemented to a final concentration of 0.25 mM, and cultures were returned to incubate overnight. Indigo production was visualized the following day by spinning down cultures and resuspending in DMSO.

Next, phage spot testing was performed to test for the generation of visibly indole-converting plaques. Briefly, 0.25 mM indole and 10 mM ara or 250 ng/μL aTc (or neither) was added to 3 mL of top agar along with 300 μL of EMG2 cells or XL10 cells overnight culture. After solidification, 7.5 μL of test phage and a 1:10 dilution series were added onto the top agar. Plates were incubated overnight at 37° C. and imaged/read one day late (Table 1).

TABLE 1 1 2 3 4 5 6 7 8 9 F 0.041 1.533 1.349 0.494 1.256 0.482 1.292 1.041 0.991 610 M9 p29 + ara p30 + aTc M29 M29 + ara Mk29 Mk29 + ara M30 M30 + aTc

This experiment revealed inducible expression of the indole-converting circuit for pBAD-styAB and plTetO-styAB constructs carried on the engineered M13 bacteriophages (FIGS. 5 and 6).

Strains:

pRJK029=MG1655Pro harboring a plasmid carrying pBAD-driven styAB;

pRJK030=MG1655Pro harboring a plasmid carrying pTet-driven styAB;

M029=EMG2 infected with M13 phage carrying pBAD-driven styAB;

M-k29=EMG2 infected with M13 phage carrying pBAD-driven styAB and a kanamycin resistance cassette; and

M030=EMG2 infected with M13 phage carrying pTet-driven styAB.

Inducers:

+ara=with 10 mM arabinose

+aTc=with 2.5 μg/mL anhydrotetracycline

M13-Indole Overnight Induction Repeat

The initial infection assay was repeated using XL10 as the recipient strain for M13 infection instead of EMG2.

The cultures were spun down at 4000 rpm for 15 min, and the pellets were resuspended in 500 μL DMSO. The resuspended pellet was transferred to 1.5 mL tubes and incubated at 60° C. with mixing for 5 min to dissolve the indigo. The dissolved indigo was spun at 10,000×g for 5 min. 300 μL of the supernatant was collected for an absorbance reading at 610 nm.

This experiment revealed inducible expression of the indole-converting circuit for both pBAD-styAB constructs carried on the engineered M13 bacteriophages. The XL10 recipient failed to show indole conversion with the M13-pTet-styAB construct, however. Since only one inducer concentration was tested, this should be optimized for M13-pTet-styAB, which was previously verified as functional using EMG2 as the recipient strain. EMG2 infected with M13-pTet-styAB looked darker than other spots when held against a white background. They looked less dark towards more concentrated phage treatment. Without being bound by theory, it is possible that the growth of bacteria is too slowed to visualize a phenotype at the higher concentrations of phage.

Example 3. Dose-Response Study of Indigo Production in Bacterial Cells Infected with Bacteriophage M029 Phage Delivery:

1. EMG2 or XL10 cells grown to stationary phase overnight. 2. Cultures washed 2× in M9 minimal media. 3. Cultures diluted 1:4 into experimental conditions. 4. Phage preps added 1:100 as well as dilutions of 10% arabinose (no inducer, 1:400, 1:200, 1:100, 1:50, 1:25, 1:12.5). 5. Cultures grown for 4 hrs at 37° C. in shaking incubator. 6. Indole added to a final concentration of 500 μM, grown for 2 hours, then assessed for Indigo production.

Indigo Collection:

1. Indigo collected from culture by spinning sample at 15 k rpm for 2 min. 2. Supernatant removed. 3. DMSO added back to recover original volume amount. 4. Pellet resuspended and mixed. 5. Samples incubated at 70° C. for 10 min to ensure all indigo dissolves into solution. 6. Samples spun down again at 15 k for 2 min. 7. 300 μL of sample added to a 96 well plate and read on a plate reader at 610 nm for OD.

FIG. 7 shows a graph of indigo concentration as a function of the increasing doses of arabinose inducer (e.g., 1:400, 1:200, 1:100, 1:50, 1:25, 1:12.5).

Example 4. Inflammatory Bowel Disease (IBD) In Vivo Model

Mice are given 2% DSS solution in place of their regular drinking water for the course of 8 days. The mice receive approximately 5 ml of DSS solution per day and the solution is replaced on days 3 and 5. After 8 days of treatment, the DSS solution is replaced with regular drinking water or drinking water that contains the therapeutic bacteriophage particles for 7 days. This protocol provides a consistent model for the inflammatory reaction of inflammatory bowel disease (Pizarro et al. Trends in Mol Med. 2003; Wirtz et al. Nature Protocols. 2007). An overview of the IBD model is shown in FIG. 5.

Example 5. Bacteriophage and Phagemid Networks In Vitro

Escherichia coli (E. coli) ER2738 bacterial cells were diluted 1:100 in media and grown for 90 minutes or until OD₆₀₀=0.2-0.3. Next, the bacterial cells were transduced with a 1:50 dilution of bacteriophage (designated M6G bacteriophage) or phagemid (designated p6T phagemid) stock. The bacteriophages and phagemids were engineered to comprise a genetic circuit that contains a nucleic acid with a constitutively active P_(TRC-2) promoter operably linked to a nucleotide sequence encoding GFP (FIG. 15A). Bacterial cells without bacteriophages and phagemids served as a negative control. The bacterial cells were then incubated with shaking at 37° C., with fluorescence measurements being taken every 2 hours by flow cytometry. Results are shown in FIG. 15B. Notably, by 6 hours, there was a 700 fold increase in GFP expression in cells transfected with the phagemids, relative to the negative control.

Example 6. Bacteriophage and Phagemid Networks In Vivo

Gnotobiotic mice (a negative control group, a positive control group and a test group) were first colonized with ER2738 E. coli bacterial cells (e.g., by spreading the bacteria on the fur of the mice and by gavage). The ER2738 E. coli bacterial cells of the positive control group contained a pLT006 plasmid carrying the genetic circuit depicted in FIG. 15A. The test group was administered 200 μl of M6G bacteriophage containing carrying the genetic circuit depicted in FIG. 15A in SM buffer via gavage one day post-colonization and three days post-colonization. The positive control group and the negative control group were given 200 μl of SM buffer only (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgSO₄)) (blank control). The mice were fed water containing tetracycline to select for ER2738 E. coli bacterial cells (which are tetracycline resistant) for a total of 5 days. On days 0 to 5, fecal samples were collected from all mice and stored at −80° C. until ready for GFP characterization. Upon GFP characterization, fecal samples were dissolved in 1 ml of phosphate buffered saline (PBS), spun down for 3 minutes at 3.4 k rpm, and analyzed for GFP expression by FACS (1,000,000 events recorded; gate set up to distinguish GFP⁺ from GFP⁻; “GFP Fluorescence geomean” and “% population GFP⁺” values recorded). Percent GFP⁺ data was used to track expression. All data scaled so that the peak GFP expression of the positive control registered as 100. This technique was used to reduce sample background. FIG. 16 shows that by day two post-colonization, 50% of the cells in the test group were positive for GFP, demonstrating successful in vivo delivery of bacteriophage engineered to contain a genetic circuit and expression of a molecule encoded by that circuit.

Methods In Vitro Protocols Phage Delivery

1. EMG2 or XL10 cells grown to stationary phase overnight. 2. Cultures washed 2× in M9 minimal media. 3. Cultures diluted 1:4 into experimental conditions. 4. Phage preps added 1:100 as well as any required inducers (e.g., 1:100 of 10% arabinose). 5. Cultures grown for 4 hours at 37° C. in shaking incubator. 6. Cultures read as needed on a fluorescence-activated cell sorting (FACS) or plate reader.

Indigo Production

1. Using the protocol described above. 2. After 4 hours of growth, indole added to a final concentration of 200-1000 μM (normal human physiological range). 3. Indole collected at desired time points as described and the concentration determined.

Indigo Collection

1. Indigo collected from culture by spinning sample at 15 k rpm for 2 min. 2. Supernatant removed. 3. DMSO added back to recover original volume amount. 4. Pellet resuspended and mixed. 5. Samples incubated at 70° C. for 10 min to ensure all indigo dissolves into solution. 6. Samples spun down again at 15 k for 2 min. 7. 300 μL of sample added to a 96 well plate and read on a plate reader at 610 nm for OD.

Cloning Methods

Standard cloning methods used, as described elsewhere herein.

In Vivo Protocols Phage Delivery Protocol

Both wild-type and gnotobiotic mice colonized with E. coli are fed bacteriophage particles carrying synthetic networks in the drinking water. Bicarbonate may be given concordantly in order to ensure phage survival through the stomach. Phage or Test networks are induced by Arabinose or aTc in the drinking water as well. Treatments are administered to mice 6 weeks or older and may be given for up to one year.

The method mentioned above may be technically challenging as mice must consume the liquid containing the bacteriophages voluntarily. As an alternative method, the bacteriophage particles (and optionally, bicarbonate) are delivered directly to (e.g., inserted directly into) the stomach through gavage. Arabinose or aTc can also be introduced into the stomach by gavage.

An enema strategy may also be used. 6-9 week old mice are first anesthetized using continuous isoflurane inhalation or intraperitoneal injection of ketamine-zylasine. Using a sterile and lubricated small gavage needle and a 1 mL syringe, 50 μL of bacteriophage particles suspended in LB or M9 minimal media are slowly injected into the colon of anesthetized mice. Air bubbles are removed from the syringe prior to the procedure. The gavage needle is inserted only deep enough to ensure the liquid remains in the colon, then the needle is withdrawn. This procedure typically takes 2-5 minutes per mouse. The infected animal is then placed back inside its cage and monitored for any signs of distress. This procedure can be done a maximum of twice a week for a maximum of 2 weeks. Any mice showing signs of severe gastrointestinal distress are euthanized.

Feces and urine (if possible) are collected from the mice and tested for signs of phage infection. Sections of the gut are examined for signs of inflammation at the end of the experiment.

Mice are monitored at least twice per week for any signs of gastrointestinal or kidney distress from the bacteriophage treatment. Mice showing signs of severe distress (hunched posture, ruffled appearance, decreased activity, etc.) are euthanized by CO₂ inhalation to effect.

An overview of the general model is shown in FIG. 6.

SEQUENCES (pRJK025) SEQ ID NO: 1 gacgtctgtgcaagtactactgttctgcagtcact tgaattcaagaaaccaatagtccatattgcatcag acattgccgtcactgcgtcttttactggctcttct cgctaaccaaaccggtaaccccgcttattaaaagc attctgtaacaaagcgggaccaaagccatgacaaa agcgcgtaacaaaagtgtctataatcacggcagaa aagtccacattgattatttgcacggcgtcacactt tgctatgccatagcatttttatccataagattagc ggatcatacctgacgctttttatcgcaactctcta ctgtttctccatacagctgaaaagcttacgggagg aacgttatgaatcagaccgacacatcacctatcag gctgcgcaggagctggaacaccagcgagatagaag cgctctttgacgagcatgccggacgtatcgatccg cgcatttataccgatgaggatctgtaccaactcga actggagcgtgtcttcgcccggtcctggctgctgt tggggcatgaaacccagattcgcaagccgggcgat tacatcacgacctacatgggtgaagaccctgtcgt ggtcgtccggcagaaagacgccagcattgccgtgt tcctgaaccagtgccgccaccgtggcatgcgcatc tgccgcgcggatgccggaaacgcgaaggcgttcac ttgcagctaccacgggtgggcttacgacaccgccg gcaatcttgtcaatgtgccttacgaggccgaatcc ttcgcgtgcctgaacaagaaggaatggagcccgct gaaggcccgggtagaaacctacaagggcctgattt tcgccaactgggatgagaacgctgtagacctcgac acgtatctgggcgaggcgaagttctacatggacca catgctcgaccgcaccgaggccggcaccgaagcga tcccgggcgtgcagaagtgggtcattccctgtaac tggaaattcgccgcagagcagttttgcagcgacat gtaccatgccgggacgacctcgcatctgtctggca tcctggcaggcctgccagaagaccttgaaatggcc gaccttgctccgccgacagttggcaagcagtaccg tgcgtcatggggcggacatggaagtggcttctatg tcggcgaccccaatctgatgcttgccatcatgggg ccaaaggtcaccagctactggaccgaaggccccgc gtcggaaaaggcggccgaacgtctgggtagcgtgg agcgcggctcgaaactcatggtcgagcacatgacc gtcttccccacgtgttccttcctcccaggtatcaa tacggtccggacatggcatccgcgcgggccgaacg aggtcgaggtatgggcgtttacggtggtcgatgct gatgctcctgacgatatcaaggaagagttccggcg ccagacgctgcgcaccttctctgccggtggcgtgt tcgagcaggacgacggggagaactgggtcgagatc cagcacatcctgcgaggccacaaggcgcggagccg ccctttcaatgccgagatgagcatggaccagaccg tcgacaacgacccggtttaccccgggcggatcagc aacaacgtctacagcgaggaagctgcccgcgggct ctatgcccattggctgcggatgatgacatcccccg actgggacgcgctgaaggcgacacgctgaatccag agacagcttgcgccacgcagtggcgccggccagag gccgcatttgacttcgacccaggttggatgcggtg gaccttgtccatttgaaatctacaaggaacgacca tgattgattcagccaacagagccgacgtctttctc cgcaagccggcacccgtagcgcccgaactgcagca cgaagtcgagcagttctactattgggaggccaagc ttctcaacgatcgccgcttcgaggagtggttcgcg ctgctcgcggaagacattcactacttcatgcccat tcgcaccacgcggatcatgcgggactcgcgccttg aatactcaggctcccgagagtacgcgcacttcgat gacgacgccacgatgatgaagggacgcttgcgcaa gatcacgtccgacgtgagctggtccgagaaccccg catcgcggacccggcatctcgtgagcaacgtgatg atcgtcggcgcagaggcagaaggggagtacgaaat ctcaagcgccttcattgtgtaccgcaatcgtctgg agcggcagctcgacatctttgccggtgagcgtcgc gatacgttgcgccgtaacacgagcgaggccgggtt cgagatcgtcaatcggaccatcctgatcgaccaga gcaccatcctggccaataacctcagtttcttcttc taggtgatgtcatgacttggacatacatattgcgg cagggtgacctgccacccggtgagatgcagcgcta cgaaggcggcccggaacctgtgatggtctgcaacg tcgatggcgagttcttcgcggtgcaggatacctgc acgcatggggactgggcgttgtcggatggttacct ggacggtgatattgtcgaatgcacgttgcatttcg gcaagttctgcgtgcggaccgggaaggtgaaggcg ctgcctgcttgcaaacctatcaaggtattcccaat caaggtcgaaggcgatgaagtgcacgtcgatctcg acaacggggagttgaagtgatggctacccatgtgg cgatcatcggcaatggcgtgggtggcttcacgacc gcgcaggccctacgtgccgagggcttcgaggggag aatctcgctgattggggacgaaccgcatctcccct atgaccgaccatccttgtccaaggcggttctcgac ggcagccttgagcggccgcccatactggccgaggc cgattggtacggcgaggcccgcatcgacatgctga ccggcccggaagtcactgcccttgatgtgcagaca aggacgatcagtctggatgatggcaccacgctctc tgcggacgccatcgtcatcgcgacgggcagtcgag cgcggacgatggcgttgcccggcagccaactgccg ggcgtcgtaacgctgcgcacctacggtgacgtgca ggtattgcgcgatagttggacttccgcgacgcggc tgctgattgtgggtggcggattgatcggctgcgag gtcgcgacgacggcgcgcaagctcggcctgtcggt cacgatcctggaggcaggtgatgaactgctggtcc gagtacttgggcggcgtatcggtgcctggctgcgc ggcctgctgacagaacttggtgtgcaggtcgagtt gggaacgggtgtcgtaggtttttctggtgagggcc agctcgaacaagtcatggccagcgatgggcgcagc ttcgtagccgatagcgcactcatttgcgtcggcgc ggagcccgcggatcaacttgcgcgtcaagcgggct tggcatgtgaccgcggcgtcattgtcgatcactgc ggtgcgacgcttgccaaaggcgtattcgccgtcgg agatgtggccagttggccgctgcgcgccggcggcc ggcgttcgctcgaaacctatatgaacgcgcagcgc caagccgccgcggtggctgcggccattctggggaa aaacgtatcggcaccgcaactgcccgtgtcctgga cggagatcgctgggcatcgcatgcagatggcgggc gatatcgaaggacctggtgatttcgtctcgcgcgg catgcccggtagtggcgctgccctgttgttccgcc tgcaggagcgaaggattcaggcggtcgtcgcggtc gatgcaccccgtgacttcgcgcttgcaacccgatt ggtagaagcccgcgcggcaatcgagccagcacggc tggcagatctttcaaacagtatgcgcgattttgtt cgtgcgaatgaaggagacctaacgtgaggtacccg agaattggcttggactcctgttgatagatccagta atgacctcagaactccatctggatttgttcagaac gctcggttgccgccgggcgttttttattggtgaga atccaagcagtagtcaggatcctcaagtcggccgc ccgttccatggatactcgtcgaccattacgctagc cgtctggagctcggactgcttaagtcgctccatat gctcgttcccgggactacacaattgtcccccggcg ccagggttgatatctatcgccctagggaccgtctc gagagaatcaatattaatccaacgcgtggcatcaa ataaaacgaaaggctcagtcgaaagactgggcctt tcgttttatctgttgtttgtcggtgaacgctctcc tgagtaggacaaatccgccgccctagacttaggcg ttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataa cgcaggaaagaacatgtgagcaaaaggccagcaaa aggccaggaaccgtaaaaaggccgcgttgctggcg tttttccataggctccgcccccctgacgagcatca caaaaatcgacgctcaagtcagaggtggcgaaacc cgacaggactataaagataccaggcgtttccccct ggaagctccctcgtgcgctctcctgttccgaccct gccgcttaccggatacctgtccgcctttctccctt cgggaagcgtggcgctttctcatagctcacgctgt aggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtctgcacgaaccccccgttcagcccg accgctgcgccttatccggtaactatcgtcttgag tccaacccggtaagacacgacttatcgccactggc agcagccactggtaacaggattagcagagcgaggt atgtaggcggtgctacagagttcttgaagtggtgg cctaactacggctacactagaaggacagtatttgg tatctgcgctctgctgaagccagttaccttcggaa aaagagttggtagctcttgatccggcaaacaaacc accgctggtagcggtggtttttttgtttgcaagca gcagattacgcgcagaaaaaaaggatctcaagaag atcctttgatcttttctacggggtctgacgctcag tggaacgaaaactcacgttaagggattttggtcat ggctagtgcttggattctcaccaataaaaaacgcc cggcggcaaccgagcgttctgaacaaatccagatg gagttctgaggtcattactggatctatcaacagga gtccaagccaattctcgaaccccagagtcccgctc agaagaactcgtcaagaaggcgatagaaggcgatg cgctgcgaatcgggagcggcgataccgtaaagcac gaggaagcggtcagcccattcgccgccaagctctt cagcaatatcacgggtagccaacgctatgtcctga tagcggtccgccacacccagccggccacagtcgat gaatccagaaaagcggccattttccaccatgatat tcggcaagcaggcatcgccgtgggtcacgacgaga tcctcgccgtcgggcatacgcgccttgagcctggc gaacagttcggctggcgcgagcccctgatgctctt cgtccagatcatcctgatcgacaagaccggcttcc atccgagtacgtgctcgctcgatgcgatgtttcgc ttggtggtcgaatgggcaggtagccggatcaagcg tatgcagccgccgcattgcatcagccatgatggat actttctcggcaggagcaaggtgagatgacaggag atcctgccccggcacttcgcccaatagcagccagt cccttcccgcttcagtgacaacgtcgagcacagcc gcgcaaggaacgcccgtcgtggccagccacgatag ccgcgctgcctcgtcttgcagttcattcagggcac cggacaggtcggtcttgacaaaaagaaccgggcga ccctgcgctgacagccggaacacggcggcatcaga gcagccgattgtctgttgtgcccagtcatagccga atagcctctccacccaagccgccggagaacctgcg tgcaatccatcttgttcaatcatgcgaaacgatcc tcatcctgtctcttgatcagatcttgatcccctgc gccatcagatccttggcggcaagaaagccatccag tttactttgcagggcttcccaaccttaccagaggg cggcccaactggcaattcc (pRJK026) SEQ ID NO: 2 gacgtctgtgcaagtactactgttctgcagtcact tgaattctccctatcagtgatagagattgacatcc ctatcagtgatagagatactgagcacatcagcagg acgcactgacccagctgaaaagcttacgggaggaa cgttatgaatcagaccgacacatcacctatcaggc tgcgcaggagctggaacaccagcgagatagaagcg ctctttgacgagcatgccggacgtatcgatccgcg catttataccgatgaggatctgtaccaactcgaac tggagcgtgtcttcgcccggtcctggctgctgttg gggcatgaaacccagattcgcaagccgggcgatta catcacgacctacatgggtgaagaccctgtcgtgg tcgtccggcagaaagacgccagcattgccgtgttc ctgaaccagtgccgccaccgtggcatgcgcatctg ccgcgcggatgccggaaacgcgaaggcgttcactt gcagctaccacgggtgggcttacgacaccgccggc aatcttgtcaatgtgccttacgaggccgaatcctt cgcgtgcctgaacaagaaggaatggagcccgctga aggcccgggtagaaacctacaagggcctgattttc gccaactgggatgagaacgctgtagacctcgacac gtatctgggcgaggcgaagttctacatggaccaca tgctcgaccgcaccgaggccggcaccgaagcgatc ccgggcgtgcagaagtgggtcattccctgtaactg gaaattcgccgcagagcagttttgcagcgacatgt accatgccgggacgacctcgcatctgtctggcatc ctggcaggcctgccagaagaccttgaaatggccga ccttgctccgccgacagttggcaagcagtaccgtg cgtcatggggcggacatggaagtggcttctatgtc ggcgaccccaatctgatgcttgccatcatggggcc aaaggtcaccagctactggaccgaaggccccgcgt cggaaaaggcggccgaacgtctgggtagcgtggag cgcggctcgaaactcatggtcgagcacatgaccgt cttccccacgtgttccttcctcccaggtatcaata cggtccggacatggcatccgcgcgggccgaacgag gtcgaggtatgggcgtttacggtggtcgatgctga tgctcctgacgatatcaaggaagagttccggcgcc agacgctgcgcaccttctctgccggtggcgtgttc gagcaggacgacggggagaactgggtcgagatcca gcacatcctgcgaggccacaaggcgcggagccgcc ctttcaatgccgagatgagcatggaccagaccgtc gacaacgacccggtttaccccgggcggatcagcaa caacgtctacagcgaggaagctgcccgcgggctct atgcccattggctgcggatgatgacatcccccgac tgggacgcgctgaaggcgacacgctgaatccagag acagcttgcgccacgcagtggcgccggccagaggc cgcatttgacttcgacccaggttggatgcggtgga ccttgtccatttgaaatctacaaggaacgaccatg attgattcagccaacagagccgacgtctttctccg caagccggcacccgtagcgcccgaactgcagcacg aagtcgagcagttctactattgggaggccaagctt ctcaacgatcgccgcttcgaggagtggttcgcgct gctcgcggaagacattcactacttcatgcccattc gcaccacgcggatcatgcgggactcgcgccttgaa tactcaggctcccgagagtacgcgcacttcgatga cgacgccacgatgatgaagggacgcttgcgcaaga tcacgtccgacgtgagctggtccgagaaccccgca tcgcggacccggcatctcgtgagcaacgtgatgat cgtcggcgcagaggcagaaggggagtacgaaatct caagcgccttcattgtgtaccgcaatcgtctggag cggcagctcgacatctttgccggtgagcgtcgcga tacgttgcgccgtaacacgagcgaggccgggttcg agatcgtcaatcggaccatcctgatcgaccagagc accatcctggccaataacctcagtttcttcttcta ggtgatgtcatgacttggacatacatattgcggca gggtgacctgccacccggtgagatgcagcgctacg aaggcggcccggaacctgtgatggtctgcaacgtc gatggcgagttcttcgcggtgcaggatacctgcac gcatggggactgggcgttgtcggatggttacctgg acggtgatattgtcgaatgcacgttgcatttcggc aagttctgcgtgcggaccgggaaggtgaaggcgct gcctgcttgcaaacctatcaaggtattcccaatca aggtcgaaggcgatgaagtgcacgtcgatctcgac aacggggagttgaagtgatggctacccatgtggcg atcatcggcaatggcgtgggtggcttcacgaccgc gcaggccctacgtgccgagggcttcgaggggagaa tctcgctgattggggacgaaccgcatctcccctat gaccgaccatccttgtccaaggcggttctcgacgg cagccttgagcggccgcccatactggccgaggccg attggtacggcgaggcccgcatcgacatgctgacc ggcccggaagtcactgcccttgatgtgcagacaag gacgatcagtctggatgatggcaccacgctctctg cggacgccatcgtcatcgcgacgggcagtcgagcg cggacgatggcgttgcccggcagccaactgccggg cgtcgtaacgctgcgcacctacggtgacgtgcagg tattgcgcgatagttggacttccgcgacgcggctg ctgattgtgggtggcggattgatcggctgcgaggt cgcgacgacggcgcgcaagctcggcctgtcggtca cgatcctggaggcaggtgatgaactgctggtccga gtacttgggcggcgtatcggtgcctggctgcgcgg cctgctgacagaacttggtgtgcaggtcgagttgg gaacgggtgtcgtaggtttttctggtgagggccag ctcgaacaagtcatggccagcgatgggcgcagctt cgtagccgatagcgcactcatttgcgtcggcgcgg agcccgcggatcaacttgcgcgtcaagcgggcttg gcatgtgaccgcggcgtcattgtcgatcactgcgg tgcgacgcttgccaaaggcgtattcgccgtcggag atgtggccagttggccgctgcgcgccggcggccgg cgttcgctcgaaacctatatgaacgcgcagcgcca agccgccgcggtggctgcggccattctggggaaaa acgtatcggcaccgcaactgcccgtgtcctggacg gagatcgctgggcatcgcatgcagatggcgggcga tatcgaaggacctggtgatttcgtctcgcgcggca tgcccggtagtggcgctgccctgttgttccgcctg caggagcgaaggattcaggcggtcgtcgcggtcga tgcaccccgtgacttcgcgcttgcaacccgattgg tagaagcccgcgcggcaatcgagccagcacggctg gcagatctttcaaacagtatgcgcgattttgttcg tgcgaatgaaggagacctaacgtgaggtacccgag aattggcttggactcctgttgatagatccagtaat gacctcagaactccatctggatttgttcagaacgc tcggttgccgccgggcgttttttattggtgagaat ccaagcagtagtcaggatcctcaagtcggccgccc gttccatggatactcgtcgaccattacgctagccg tctggagctcggactgcttaagtcgctccatatgc tcgttcccgggactacacaattgtcccccggcgcc agggttgatatctatcgccctagggaccgtctcga gagaatcaatattaatccaacgcgtggcatcaaat aaaacgaaaggctcagtcgaaagactgggcctttc gttttatctgttgtttgtcggtgaacgctctcctg agtaggacaaatccgccgccctagacttaggcgtt cggctgcggcgagcggtatcagctcactcaaaggc ggtaatacggttatccacagaatcaggggataacg caggaaagaacatgtgagcaaaaggccagcaaaag gccaggaaccgtaaaaaggccgcgttgctggcgtt tttccataggctccgcccccctgacgagcatcaca aaaatcgacgctcaagtcagaggtggcgaaacccg acaggactataaagataccaggcgtttccccctgg aagctccctcgtgcgctctcctgttccgaccctgc cgcttaccggatacctgtccgcctttctcccttcg ggaagcgtggcgctttctcatagctcacgctgtag gtatctcagttcggtgtaggtcgttcgctccaagc tgggctgtctgcacgaaccccccgttcagcccgac cgctgcgccttatccggtaactatcgtcttgagtc caacccggtaagacacgacttatcgccactggcag cagccactggtaacaggattagcagagcgaggtat gtaggcggtgctacagagttcttgaagtggtggcc taactacggctacactagaaggacagtatttggta tctgcgctctgctgaagccagttaccttcggaaaa agagttggtagctcttgatccggcaaacaaaccac cgctggtagcggtggttatttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatc ctttgatcttttctacggggtctgacgctcagtgg aacgaaaactcacgttaagggattttggtcatggc tagtgcttggattctcaccaataaaaaacgcccgg cggcaaccgagcgttctgaacaaatccagatggag ttctgaggtcattactggatctatcaacaggagtc caagccaattctcgaaccccagagtcccgctcaga agaactcgtcaagaaggcgatagaaggcgatgcgc tgcgaatcgggagcggcgataccgtaaagcacgag gaagcggtcagcccattcgccgccaagctcttcag caatatcacgggtagccaacgctatgtcctgatag cggtccgccacacccagccggccacagtcgatgaa tccagaaaagcggccattttccaccatgatattcg gcaagcaggcatcgccgtgggtcacgacgagatcc tcgccgtcgggcatacgcgccttgagcctggcgaa cagttcggctggcgcgagcccctgatgctcttcgt ccagatcatcctgatcgacaagaccggcttccatc cgagtacgtgctcgctcgatgcgatgtttcgcttg gtggtcgaatgggcaggtagccggatcaagcgtat gcagccgccgcattgcatcagccatgatggatact ttctcggcaggagcaaggtgagatgacaggagatc ctgccccggcacttcgcccaatagcagccagtccc ttcccgcttcagtgacaacgtcgagcacagccgcg caaggaacgcccgtcgtggccagccacgatagccg cgctgcctcgtcttgcagttcattcagggcaccgg acaggtcggtcttgacaaaaagaaccgggcgaccc tgcgctgacagccggaacacggcggcatcagagca gccgattgtctgttgtgcccagtcatagccgaata gcctctccacccaagccgccggagaacctgcgtgc aatccatcttgttcaatcatgcgaaacgatcctca tcctgtctcttgatcagatcttgatcccctgcgcc atcagatccttggcggcaagaaagccatccagttt actttgcagggcttcccaaccttaccagagggcgg cccaactggcaattcc (pRJK029) SEQ ID NO: 3 gacgtctgtgcaagtactaagaaaccaatagtcca tattgcatcagacattgccgtcactgcgtctttta ctggctcttctcgctaaccaaaccggtaaccccgc ttattaaaagcattctgtaacaaagcgggaccaaa gccatgacaaaagcgcgtaacaaaagtgtctataa tcacggcagaaaagtccacattgattatttgcacg gcgtcacactttgctatgccatagcatttttatcc ataagattagcggatcatacctgacgctttttatc gcaactctctactgtttctccatacagctgaagga ttaaggaggtagcatgcatgaaaaagcgtatcggt attgttggtgcaggcactgccggcctccatcttgg cctcttcctccgccagcatgacgtcgacgtcactg tgtacactgatcgtaagcccgatgagtacagtgga ctgcggctcctgaataccgttgctcacaacgcggt gacggtgcagcgggaggttgccctcgacgtcaatg agtggccgtctgaggagtttggctatttcggccac tactactacgtaggtgggccgcagcccatgcgttt ctacggtgatctcaaggctcccagccgtgcagtgg actaccgtctctacctgccgatgctgatgcgtgca ctggaagccaggggcggcaagttctgctacgacgc cgtgtctgccgaagatctggaagggctgtcggagc agtatgatctgctggttgtgtgcactggtaaatac gccctcggcaaggtgttcgagaagcagtccgaaaa ctcgcccttcgagaagccgcaacgggcactgtgcg ttggtctcttcaagggcatcaaggaagcaccgatt cgcgcggtgactatgtccttctcgccagggcatgg cgagctgattgagattccaaccctgtcgttcaatg gcatgagcacagcgctggtgctcgaaaaccatatt ggtagcgatctggaagtcctcgcccacaccaagta tgacgatgacccgcgtgcgttcctcgatctgatgc tggagaagctgcgtaagcatcatccttccgttgcc gagcgcatcgatccggctgagttcgacctggccaa cagttctctggacatcctccagggcggtgttgtgc cagtattccgcgacggtcatgcgaccctcaataac ggcaaaaccatcatcgggctgggcgacatccaggc aactgtcgatccggtcttgggccagggcgcgaaca tggcgtcctatgcggcatggattctgggcgaggaa atccttgcgcactctgtctacgacctgcgcttcag cgaacacctggagcgtcgccgccaggatcgcgtgc tgtgcgccacccgctggaccaacttcactctgagc gccttcacggaacttccgccggaattcctcacctt ccttcagatcctgagccagagccgtgaaatggctg atgagttcacggacaacttcaactatccggaactt cagtgggatcgcttctccagcccggaacgtatcgg tcagtggtgcagccagtacgcacccactattgcgg cctgacgctattgctccgctggtcaaggccagcgg agccctaactcctgggtgattcaaatgacgttaaa aaaagatgtggtggtggatatcgactccaccagct tccgccaggcggttgcactgttcgcgacgggaatt gcggttctcagcgcggagactgacgagggcgaagt gcatggcatgacggtgaacagcttcacctccatca gtctggacccgccgactgtgatggtgtccctgaag tcgggccgtatgcatgagctgctgactcaaggcgg acgcttcggcgtcagcctcctgggtgaaagtcaga agatgttatcggcattcttcagcaagcgtgtgatc gatggcactcctcctcctgctttcacagttcaggc cggcctccccactctgcgggacgccatggcctggt tcgaatgcgaggtggagagcacggttgaagtacac gaccacacgctcttcattgcgcgcgttagcgcctg tggagtgccggaggcgaatgccccccagccgctgc tgttctttgccagccgttatcacggcaacccgttg ccgctgaattgaaacgttcgagaattggcttggac tcctgttgatagatccagtaatgacctcagaactc catctggatttgttcagaacgctcggttgccgccg ggcgttttttattggtgagaatccaagcagtagtc aaagcttccgcaaggtaccactttgccgcggagta tttgtacatttgaaggatcctcaagtcggccgccc gttccatggatactcgtcgaccattacgctagccg tctggagctcggactgcttaagtcgctccatatgc tcgttcccgggactacacaattgtcccccggcgcc agggttgatatctatcgccctagggaccgtctcga gagaatcaatattaatccaacgcgtggcatcaaat aaaacgaaaggctcagtcgaaagactgggcctttc gttttatctgttgtttgtcggtgaacgctctcctg agtaggacaaatccgccgccctagacttaggcgtt cggctgcggcgagcggtatcagctcactcaaaggc ggtaatacggttatccacagaatcaggggataacg caggaaagaacatgtgagcaaaaggccagcaaaag gccaggaaccgtaaaaaggccgcgttgctggcgtt tttccataggctccgcccccctgacgagcatcaca aaaatcgacgctcaagtcagaggtggcgaaacccg acaggactataaagataccaggcgtttccccctgg aagctccctcgtgcgctctcctgttccgaccctgc cgcttaccggatacctgtccgcctttctcccttcg ggaagcgtggcgctttctcatagctcacgctgtag gtatctcagttcggtgtaggtcgttcgctccaagc tgggctgtctgcacgaaccccccgttcagcccgac cgctgcgccttatccggtaactatcgtcttgagtc caacccggtaagacacgacttatcgccactggcag cagccactggtaacaggattagcagagcgaggtat gtaggcggtgctacagagttcttgaagtggtggcc taactacggctacactagaaggacagtatttggta tctgcgctctgctgaagccagttaccttcggaaaa agagttggtagctcttgatccggcaaacaaaccac cgctggtagcggtggttatttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatc ctttgatcttttctacggggtctgacgctcagtgg aacgaaaactcacgttaagggattttggtcatggc tagtgcttggattctcaccaataaaaaacgcccgg cggcaaccgagcgttctgaacaaatccagatggag ttctgaggtcattactggatctatcaacaggagtc caagccaattctcgaaccccagagtcccgctcaga agaactcgtcaagaaggcgatagaaggcgatgcgc tgcgaatcgggagcggcgataccgtaaagcacgag gaagcggtcagcccattcgccgccaagctcttcag caatatcacgggtagccaacgctatgtcctgatag cggtccgccacacccagccggccacagtcgatgaa tccagaaaagcggccattttccaccatgatattcg gcaagcaggcatcgccgtgggtcacgacgagatcc tcgccgtcgggcatacgcgccttgagcctggcgaa cagttcggctggcgcgagcccctgatgctcttcgt ccagatcatcctgatcgacaagaccggcttccatc cgagtacgtgctcgctcgatgcgatgtttcgcttg gtggtcgaatgggcaggtagccggatcaagcgtat gcagccgccgcattgcatcagccatgatggatact ttctcggcaggagcaaggtgagatgacaggagatc ctgccccggcacttcgcccaatagcagccagtccc ttcccgcttcagtgacaacgtcgagcacagccgcg caaggaacgcccgtcgtggccagccacgatagccg cgctgcctcgtcttgcagttcattcagggcaccgg acaggtcggtcttgacaaaaagaaccgggcgaccc tgcgctgacagccggaacacggcggcatcagagca gccgattgtctgttgtgcccagtcatagccgaata gcctctccacccaagccgccggagaacctgcgtgc aatccatcttgttcaatcatgcgaaacgatcctca tcctgtctcttgatcagatcttgatcccctgcgcc atcagatccttggcggcaagaaagccatccagttt actttgcagggcttcccaaccttaccagagggcgg cccaactggcaattcc (pRJK030) SEQ ID NO: 4 gacgtctgtgcaagtacttccctatcagtgataga gattgacatccctatcagtgatagagatactgagc acatcagcaggacgcactgacccagctgaaggatt aaggaggtagcatgcatgaaaaagcgtatcggtat tgttggtgcaggcactgccggcctccatcttggcc tcttcctccgccagcatgacgtcgacgtcactgtg tacactgatcgtaagcccgatgagtacagtggact gcggctcctgaataccgttgctcacaacgcggtga cggtgcagcgggaggttgccctcgacgtcaatgag tggccgtctgaggagtttggctatttcggccacta ctactacgtaggtgggccgcagcccatgcgtttct acggtgatctcaaggctcccagccgtgcagtggac taccgtctctacctgccgatgctgatgcgtgcact ggaagccaggggcggcaagttctgctacgacgccg tgtctgccgaagatctggaagggctgtcggagcag tatgatctgctggttgtgtgcactggtaaatacgc cctcggcaaggtgttcgagaagcagtccgaaaact cgcccttcgagaagccgcaacgggcactgtgcgtt ggtctcttcaagggcatcaaggaagcaccgattcg cgcggtgactatgtccttctcgccagggcatggcg agctgattgagattccaaccctgtcgttcaatggc atgagcacagcgctggtgctcgaaaaccatattgg tagcgatctggaagtcctcgcccacaccaagtatg acgatgacccgcgtgcgttcctcgatctgatgctg gagaagctgcgtaagcatcatccttccgttgccga gcgcatcgatccggctgagttcgacctggccaaca gttctctggacatcctccagggcggtgttgtgcca gtattccgcgacggtcatgcgaccctcaataacgg caaaaccatcatcgggctgggcgacatccaggcaa ctgtcgatccggtcttgggccagggcgcgaacatg gcgtcctatgcggcatggattctgggcgaggaaat ccttgcgcactctgtctacgacctgcgcttcagcg aacacctggagcgtcgccgccaggatcgcgtgctg tgcgccacccgctggaccaacttcactctgagcgc cttcacggaacttccgccggaattcctcaccttcc ttcagatcctgagccagagccgtgaaatggctgat gagttcacggacaacttcaactatccggaacttca gtgggatcgcttctccagcccggaacgtatcggtc agtggtgcagccagtacgcacccactattgcggcc tgacgctattgctccgctggtcaaggccagcggag ccctaactcctgggtgattcaaatgacgttaaaaa aagatgtggtggtggatatcgactccaccagcttc cgccaggcggttgcactgttcgcgacgggaattgc ggttctcagcgcggagactgacgagggcgaagtgc atggcatgacggtgaacagcttcacctccatcagt ctggacccgccgactgtgatggtgtccctgaagtc gggccgtatgcatgagctgctgactcaaggcggac gcttcggcgtcagcctcctgggtgaaagtcagaag atgttatcggcattcttcagcaagcgtgtgatcga tggcactcctcctcctgctttcacagttcaggccg gcctccccactctgcgggacgccatggcctggttc gaatgcgaggtggagagcacggttgaagtacacga ccacacgctcttcattgcgcgcgttagcgcctgtg gagtgccggaggcgaatgccccccagccgctgctg ttctttgccagccgttatcacggcaacccgttgcc gctgaattgaaacgttcgagaattggcttggactc ctgttgatagatccagtaatgacctcagaactcca tctggatttgttcagaacgctcggttgccgccggg cgttttttattggtgagaatccaagcagtagtcaa agcttccgcaaggtaccactttgccgcggagtatt tgtacatttgaaggatcctcaagtcggccgcccgt tccatggatactcgtcgaccattacgctagccgtc tggagctcggactgcttaagtcgctccatatgctc gttcccgggactacacaattgtcccccggcgccag ggttgatatctatcgccctagggaccgtctcgaga gaatcaatattaatccaacgcgtggcatcaaataa aacgaaaggctcagtcgaaagactgggcctttcgt tttatctgttgtttgtcggtgaacgctctcctgag taggacaaatccgccgccctagacttaggcgttcg gctgcggcgagcggtatcagctcactcaaaggcgg taatacggttatccacagaatcaggggataacgca ggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttt tccataggctccgcccccctgacgagcatcacaaa aatcgacgctcaagtcagaggtggcgaaacccgac aggactataaagataccaggcgtttccccctggaa gctccctcgtgcgctctcctgttccgaccctgccg cttaccggatacctgtccgcctttctcccttcggg aagcgtggcgctttctcatagctcacgctgtaggt atctcagttcggtgtaggtcgttcgctccaagctg ggctgtctgcacgaaccccccgttcagcccgaccg ctgcgccttatccggtaactatcgtcttgagtcca acccggtaagacacgacttatcgccactggcagca gccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggccta actacggctacactagaaggacagtatttggtatc tgcgctctgctgaagccagttaccttcggaaaaag agttggtagctcttgatccggcaaacaaaccaccg ctggtagcggtggttatttgtttgcaagcagcaga ttacgcgcagaaaaaaaggatctcaagaagatcct ttgatcttttctacggggtctgacgctcagtggaa cgaaaactcacgttaagggattttggtcatggcta gtgcttggattctcaccaataaaaaacgcccggcg gcaaccgagcgttctgaacaaatccagatggagtt ctgaggtcattactggatctatcaacaggagtcca agccaattctcgaaccccagagtcccgctcagaag aactcgtcaagaaggcgatagaaggcgatgcgctg cgaatcgggagcggcgataccgtaaagcacgagga agcggtcagcccattcgccgccaagctcttcagca atatcacgggtagccaacgctatgtcctgatagcg gtccgccacacccagccggccacagtcgatgaatc cagaaaagcggccattttccaccatgatattcggc aagcaggcatcgccgtgggtcacgacgagatcctc gccgtcgggcatacgcgccttgagcctggcgaaca gttcggctggcgcgagcccctgatgctcttcgtcc agatcatcctgatcgacaagaccggcttccatccg agtacgtgctcgctcgatgcgatgtttcgcttggt ggtcgaatgggcaggtagccggatcaagcgtatgc agccgccgcattgcatcagccatgatggatacttt ctcggcaggagcaaggtgagatgacaggagatcct gccccggcacttcgcccaatagcagccagtccctt cccgcttcagtgacaacgtcgagcacagccgcgca aggaacgcccgtcgtggccagccacgatagccgcg ctgcctcgtcttgcagttcattcagggcaccggac aggtcggtcttgacaaaaagaaccgggcgaccctg cgctgacagccggaacacggcggcatcagagcagc cgattgtctgttgtgcccagtcatagccgaatagc ctctccacccaagccgccggagaacctgcgtgcaa tccatcttgttcaatcatgcgaaacgatcctcatc ctgtctcttgatcagatcttgatcccctgcgccat cagatccttggcggcaagaaagccatccagtttac tttgcagggcttcccaaccttaccagagggcggcc caactggcaattcc (M029) SEQ ID NO: 5 aatgctactactattagtagaattgatgccacctt ttcagctcgcgccccaaatgaaaatatagctaaac aggttattgaccatttgcgaaatgtatctaatggt caaactaaatctactcgttcgcagaattgggaatc aactgttatatggaatgaaacttccagacaccgta ctttagttgcatatttaaaacatgttgagctacag cattatattcagcaattaagctctaagccatccgc aaaaatgacctcttatcaaaaggagcaattaaagg tactctctaatcctgacctgttggagtttgcttcc ggtctggttcgctttgaagctcgaattaaaacgcg atatttgaagtctttcgggcttcctcttaatcttt ttgatgcaatccgctttgcttctgactataatagt cagggtaaagacctgatttttgatttatggtcatt ctcgttttctgaactgtttaaagcatttgaggggg attcaatgaatatttatgacgattccgcagtattg gacgctatccagtctaaacattttactattacccc ctctggcaaaacttcttttgcaaaagcctctcgct attttggtttttatcgtcgtctggtaaacgagggt tatgatagtgttgctcttactatgcctcgtaattc cttttggcgttatgtatctgcattagttgaatgtg gtattcctaaatctcaactgatgaatctttctacc tgtaataatgttgttccgttagttcgttttattaa cgtagatttttcttcccaacgtcctgactggtata atgagccagttcttaaaatcgcataaggtaattca caatgattaaagttgaaattaaaccatctcaagcc caatttactactcgttctggtgtttctcgtcaggg caagccttattcactgaatgagcagctttgttacg ttgatttgggtaatgaatatccggttcttgtcaag attactcttgatgaaggtcagccagcctatgcgcc tggtctgtacaccgttcatctgtcctctttcaaag ttggtcagttcggttcccttatgattgaccgtctg cgcctcgttccggctaagtaacatggagcaggtcg cggatttcgacacaatttatcaggcgatgatacaa atctccgttgtactttgtttcgcgcttggtataat cgctgggggtcaaagatgagtgttttagtgtattc ttttgcctctttcgttttaggttggtgccttcgta gtggcattacgtattttacccgtttaatggaaact tcctcatgaaaaagtctttagtcctcaaagcctct gtagccgttgctaccctcgttccgatgctgtcttt cgctgctgagggtgacgatcccgcaaaagcggcct ttaactccctgcaagcctcagcgaccgaatatatc ggttatgcgtgggcgatggttgttgtcattgtcgg cgcaactatcggtatcaagctgtttaagaaattca cctcgaaagcaagctgataaaccgatacaattaaa ggctccttttggagccttttttttggagattttca acgtgaaaaaattattattcgcaattcctttagtt gttcctttctattctcactccgctgaaactgttga aagttgtttagcaaaatcccatacagaaaattcat ttactaacgtctggaaagacgacaaaactttagat cgttacgctaactatgagggctgtctgtggaatgc tacaggcgttgtagtttgtactggtgacgaaactc agtgttacggtacatgggttcctattgggcttgct atccctgaaaatgagggtggtggctctgagggtgg cggttctgagggtggcggttctgagggtggcggta ctaaacctcctgagtacggtgatacacctattccg ggctatacttatatcaaccctctcgacggcactta tccgcctggtactgagcaaaaccccgctaatccta atccttctcttgaggagtctcagcctcttaatact ttcatgtttcagaataataggttccgaaataggca gggggcattaactgtttatacgggcactgttactc aaggcactgaccccgttaaaacttattaccagtac actcctgtatcatcaaaagccatgtatgacgctta ctggaacggtaaattcagagactgcgctttccatt ctggctttaatgaggatttatttgtttgtgaatat caaggccaatcgtctgacctgcctcaacctcctgt caatgctggcggcggctctggtggtggttctggtg gcggctctgagggtggtggctctgagggtggcggt tctgagggtggcggctctgagggaggcggttccgg tggtggctctggttccggtgattttgattatgaaa agatggcaaacgctaataagggggctatgaccgaa aatgccgatgaaaacgcgctacagtctgacgctaa aggcaaacttgattctgtcgctactgattacggtg ctgctatcgatggtttcattggtgacgtttccggc cttgctaatggtaatggtgctactggtgattttgc tggctctaattcccaaatggctcaagtcggtgacg gtgataattcacctttaatgaataatttccgtcaa tatttaccttccctccctcaatcggttgaatgtcg cccttttgtctttggcgctggtaaaccatatgaat tttctattgattgtgacaaaataaacttattccgt ggtgtctttgcgtttcttttatatgttgccacctt tatgtatgtattttctacgtttgctaacatactgc gtaataaggagtcttaatcatgccagttcttttgg gtattccgttattattgcgtttcctcggtttcctt ctggtaactttgttcggctatctgcttacttttct taaaaagggcttcggtaagatagctattgctattt cattgtttcttgctcttattattgggcttaactca attcttgtgggttatctctctgatattagcgctca attaccctctgactttgttcagggtgttcagttaa ttctcccgtctaatgcgcttccctgtttttatgtt attctctctgtaaaggctgctattttcatttttga cgttaaacaaaaaatcgtttcttatttggattggg ataaataatatggctgtttattttgtaactggcaa attaggctctggaaagacgctcgttagcgttggta agattcaggataaaattgtagctgggtgcaaaata gcaactaatcttgatttaaggcttcaaaacctccc gcaagtcgggaggttcgctaaaacgcctcgcgttc ttagaataccggataagccttctatatctgatttg cttgctattgggcgcggtaatgattcctacgatga aaataaaaacggcttgcttgttctcgatgagtgcg gtacttggtttaatacccgttcttggaatgataag gaaagacagccgattattgattggtttctacatgc tcgtaaattaggatgggatattatttttcttgttc aggacttatctattgttgataaacaggcgcgttct gcattagctgaacatgttgtttattgtcgtcgtct ggacagaattactttaccttttgtcggtactttat attctcttattactggctcgaaaatgcctctgcct aaattacatgttggcgttgttaaatatggcgattc tcaattaagccctactgttgagcgttggctttata ctggtaagaatttgtataacgcatatgatactaaa caggctttttctagtaattatgattccggtgttta ttcttatttaacgccttatttatcacacggtcggt atttcaaaccattaaatttaggtcagaagatgaaa ttaactaaaatatatttgaaaaagttttctcgcgt tctttgtcttgcgattggatttgcatcagcattta catatagttatataacccaacctaagccggaggtt aaaaaggtagtctctcagacctatgattttgataa attcactattgactcttctcagcgtcttaatctaa gctatcgctatgttttcaaggattctaagggaaaa ttaattaatagcgacgatttacagaagcaaggtta ttcactcacatatattgatttatgtactgtttcca ttaaaaaaggtaattcaaatgaaattgttaaatgt aattaattttgattcttgatgtttgtttcatcatc ttcttttgctcaggtaattgaaatgaataattcgc ctctgcgcgattttgtaacttggtattcaaagcaa tcaggcgaatccgttattgtttctcccgatgtaaa aggtactgttactgtatattcatctgacgttaaac ctgaaaatctacgcaatttctttatttctgtttta cgtgcaaataattttgatatggtaggttctaaccc ttccattattcagaagtataatccaaacaatcagg attatattgatgaattgccatcatctgataatcag gaatatgatgataattccgctccttctggtggttt ctttgttccgcaaaatgataatgttactcaaactt ttaaaattaataacgttcgggcaaaggatttaata cgagttgtcgaattgtttgtaaagtctaatacttc taaatcctcaaatgtattatctattgacggctcta atctattagttgttagtgctcctaaagatatttta gataaccttcctcaattcctttcaactgttgattt gccaactgaccagatattgattgagggtttgatat ttgaggttcagcaaggtgatgctttagatttttca tttgctgctggctctcagcgtggcactgttgcagg cggtgttaatactgaccgcctcacctctgattatc ttctgctggtggttcgttcggtatttttaatggcg atgattagggctatcagttcgcgcattaaagacta atagccattcaaaaatattgtctgtgccacgtatt cttacgctttcaggtcagaagggttctatctctgt tggccagaatgtcccttttattactggtcgtgtga ctggtgaatctgccaatgtaaataatccatttcag acgattgagcgtcaaaatgtaggtatttccatgag cgtttttcctgttgcaatggctggcggtaatattg ttctggatattaccagcaaggccgatagtttgagt tcttctactcaggcaagtgatgttattactaatca aagaagtattgctacaacggttaatttgcgtgatg gacagactcttttactcggtggcctcactgattat aaaaacacttctcaggattctggcgtaccgttcct gtctaaaatccctttaatcggcctcctgtttagct cccgctctgattctaacgaggaaagcacgttatac gtgctcgtcaaagcaaccatagtacgcgccctgta gcggcgcattaagcgcggcgggtgtggtggttacg cgcagcgtgaccgctacacttgccagcgccctagc gcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaat cgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgatttgggtg atggttcacgtagtgggccatcgccctgatagacg gtttttcgccctttgacgttggagtccacgttctt taatagtggactcttgttccaaactggaacaacac tcaaccctatctcgggctattcttttgatttataa gggattttgccgatttcggaaccaccatcaaacag gattttcgcctgctggggcaaaccagcgtggaccg cttgctgcaactctctcagggccaggcggtgaagg gcaatcagctgttgcccgtctcactggtgaaaaga aaaaccaccctggcgcccaatacgcaaaccgcctc tccccgcgcgttggccgattcattaatgcagctgg cacgacaggtttcccgactggaaagcgggcagtga gcgcaacgcaattaatgtgagttagctcactcatt aggcaccccaggctttacactttatgcttccggct cgtatgttgtgtggaattgtgagcggataacaatt tcacacaggaaacagctatgaccatgattacgaat tcgagctctgactactgcttggattctcaccaata aaaaacgcccggcggcaaccgagcgttctgaacaa atccagatggagttctgaggtcattactggatcta tcaacaggagtccaagccaattctcgaacgtttca attcagcggcaacgggttgccgtgataacggctgg caaagaacagcagcggctggggggcattcgcctcc ggcactccacaggcgctaacgcgcgcaatgaagag cgtgtggtcgtgtacttcaaccgtgctctccacct cgcattcgaaccaggccatggcgtcccgcagagtg gggaggccggcctgaactgtgaaagcaggaggagg agtgccatcgatcacacgcttgctgaagaatgccg ataacatcttctgactttcacccaggaggctgacg ccgaagcgtccgccttgagtcagcagctcatgcat acggcccgacttcagggacaccatcacagtcggcg ggtccagactgatggaggtgaagctgttcaccgtc atgccatgcacttcgccctcgtcagtctccgcgct gagaaccgcaattcccgtcgcgaacagtgcaaccg cctggcggaagctggtggagtcgatatccaccacc acatctttttttaacgtcatttgaatcacccagga gttagggctccgctggccttgaccagcggagcaat agcgtcaggccgcaatagtgggtgcgtactggctg caccactgaccgatacgttccgggctggagaagcg atcccactgaagttccggatagttgaagttgtccg tgaactcatcagccatttcacggctctggctcagg atctgaaggaaggtgaggaattccggcggaagttc cgtgaaggcgctcagagtgaagttggtccagcggg tggcgcacagcacgcgatcctggcggcgacgctcc aggtgttcgctgaagcgcaggtcgtagacagagtg cgcaaggatttcctcgcccagaatccatgccgcat aggacgccatgttcgcgccctggcccaagaccgga tcgacagttgcctggatgtcgcccagcccgatgat ggttttgccgttattgagggtcgcatgaccgtcgc ggaatactggcacaacaccgccctggaggatgtcc agagaactgttggccaggtcgaactcagccggatc gatgcgctcggcaacggaaggatgatgcttacgca gcttctccagcatcagatcgaggaacgcacgcggg tcatcgtcatacttggtgtgggcgaggacttccag atcgctaccaatatggttttcgagcaccagcgctg tgctcatgccattgaacgacagggttggaatctca atcagctcgccatgccctggcgagaaggacatagt caccgcgcgaatcggtgcttccttgatgcccttga agagaccaacgcacagtgcccgttgcggcttctcg aagggcgagttttcggactgcttctcgaacacctt gccgagggcgtatttaccagtgcacacaaccagca gatcatactgctccgacagcccttccagatcttcg gcagacacggcgtcgtagcagaacttgccgcccct ggcttccagtgcacgcatcagcatcggcaggtaga gacggtagtccactgcacggctgggagccttgaga tcaccgtagaaacgcatgggctgcggcccacctac gtagtagtagtggccgaaatagccaaactcctcag acggccactcattgacgtcgagggcaacctcccgc tgcaccgtcaccgcgttgtgagcaacggtattcag gagccgcagtccactgtactcatcgggcttacgat cagtgtacacagtgacgtcgacgtcatgctggcgg aggaagaggccaagatggaggccggcagtgcctgc accaacaataccgatacgctttttcatgcatgcta cctccttaatccttcagctgtatggagaaacagta gagagttgcgataaaaagcgtcaggtatgatccgc taatcttatggataaaaatgctatggcatagcaaa gtgtgacgccgtgcaaataatcaatgtggactttt ctgccgtgattatagacacttttgttacgcgcttt tgtcatggctttggtcccgctttgttacagaatgc ttttaataagcggggttaccggtttggttagcgag aagagccagtaaaagacgcagtgacggcaatgtct gatgcaatatggactattggtttcttggtacccgg ggatcctctagagtcgacctgcaggcatgcaagct tggcactggccgtcgttttacaacgtcgtgactgg gaaaaccctggcgttacccaacttaatcgccttgc agcacatccccctttcgccagctggcgtaatagcg aagaggcccgcaccgatcgcccttcccaacagttg cgcagcctgaatggcgaatggcgctttgcctggtt tccggcaccagaagcggtgccggaaagctggctgg agtgcgatcttcctgaggccgatactgtcgtcgtc ccctcaaactggcagatgcacggttacgatgcgcc catctacaccaacgtgacctatcccattacggtca atccgccgtttgttcccacggagaatccgacgggt tgttactcgctcacatttaatgttgatgaaagctg gctacaggaaggccagacgcgaattatttttgatg gcgttcctattggttaaaaaatgagctgatttaac aaaaatttaatgcgaattttaacaaaatattaacg tttacaatttaaatatttgcttatacaatcttcct gtttttggggcttttctgattatcaaccggggtac atatgattgacatgctagttttacgattaccgttc atcgattctcttgtttgctccagactctcaggcaa tgacctgatagcctttgtagatctctcaaaaatag ctaccctctccggcattaatttatcagctagaacg gttgaatatcatattgatggtgatttgactgtctc cggcctttctcacccttttgaatctttacctacac attactcaggcattgcatttaaaatatatgagggt tctaaaaatttttatccttgcgttgaaataaaggc ttctcccgcaaaagtattacagggtcataatgatt tggtacaaccgatttagctttatgctctgaggctt tattgcttaattttgctaattctttgccttgcctg tatgatttattggatgtt (M030) SEQ ID NO: 6 aatgctactactattagtagaattgatgccacctt ttcagctcgcgccccaaatgaaaatatagctaaac aggttattgaccatttgcgaaatgtatctaatggt caaactaaatctactcgttcgcagaattgggaatc aactgttatatggaatgaaacttccagacaccgta ctttagttgcatatttaaaacatgttgagctacag cattatattcagcaattaagctctaagccatccgc aaaaatgacctcttatcaaaaggagcaattaaagg tactctctaatcctgacctgttggagtttgcttcc ggtctggttcgctttgaagctcgaattaaaacgcg atatttgaagtctttcgggcttcctcttaatcttt ttgatgcaatccgctttgcttctgactataatagt cagggtaaagacctgatttttgatttatggtcatt ctcgttttctgaactgtttaaagcatttgaggggg attcaatgaatatttatgacgattccgcagtattg gacgctatccagtctaaacattttactattacccc ctctggcaaaacttcttttgcaaaagcctctcgct attttggtttttatcgtcgtctggtaaacgagggt tatgatagtgttgctcttactatgcctcgtaattc cttttggcgttatgtatctgcattagttgaatgtg gtattcctaaatctcaactgatgaatctttctacc tgtaataatgttgttccgttagttcgttttattaa cgtagatttttcttcccaacgtcctgactggtata atgagccagttcttaaaatcgcataaggtaattca caatgattaaagttgaaattaaaccatctcaagcc caatttactactcgttctggtgtttctcgtcaggg caagccttattcactgaatgagcagctttgttacg ttgatttgggtaatgaatatccggttcttgtcaag attactcttgatgaaggtcagccagcctatgcgcc tggtctgtacaccgttcatctgtcctctttcaaag ttggtcagttcggttcccttatgattgaccgtctg cgcctcgttccggctaagtaacatggagcaggtcg cggatttcgacacaatttatcaggcgatgatacaa atctccgttgtactttgtttcgcgcttggtataat cgctgggggtcaaagatgagtgttttagtgtattc ttttgcctctttcgttttaggttggtgccttcgta gtggcattacgtattttacccgtttaatggaaact tcctcatgaaaaagtctttagtcctcaaagcctct gtagccgttgctaccctcgttccgatgctgtcttt cgctgctgagggtgacgatcccgcaaaagcggcct ttaactccctgcaagcctcagcgaccgaatatatc ggttatgcgtgggcgatggttgttgtcattgtcgg cgcaactatcggtatcaagctgtttaagaaattca cctcgaaagcaagctgataaaccgatacaattaaa ggctccttttggagccttttttttggagattttca acgtgaaaaaattattattcgcaattcctttagtt gttcctttctattctcactccgctgaaactgttga aagttgtttagcaaaatcccatacagaaaattcat ttactaacgtctggaaagacgacaaaactttagat cgttacgctaactatgagggctgtctgtggaatgc tacaggcgttgtagtttgtactggtgacgaaactc agtgttacggtacatgggttcctattgggcttgct atccctgaaaatgagggtggtggctctgagggtgg cggttctgagggtggcggttctgagggtggcggta ctaaacctcctgagtacggtgatacacctattccg ggctatacttatatcaaccctctcgacggcactta tccgcctggtactgagcaaaaccccgctaatccta atccttctcttgaggagtctcagcctcttaatact ttcatgtttcagaataataggttccgaaataggca gggggcattaactgtttatacgggcactgttactc aaggcactgaccccgttaaaacttattaccagtac actcctgtatcatcaaaagccatgtatgacgctta ctggaacggtaaattcagagactgcgctttccatt ctggctttaatgaggatttatttgtttgtgaatat caaggccaatcgtctgacctgcctcaacctcctgt caatgctggcggcggctctggtggtggttctggtg gcggctctgagggtggtggctctgagggtggcggt tctgagggtggcggctctgagggaggcggttccgg tggtggctctggttccggtgattttgattatgaaa agatggcaaacgctaataagggggctatgaccgaa aatgccgatgaaaacgcgctacagtctgacgctaa aggcaaacttgattctgtcgctactgattacggtg ctgctatcgatggtttcattggtgacgtttccggc cttgctaatggtaatggtgctactggtgattttgc tggctctaattcccaaatggctcaagtcggtgacg gtgataattcacctttaatgaataatttccgtcaa tatttaccttccctccctcaatcggttgaatgtcg cccttttgtctttggcgctggtaaaccatatgaat tttctattgattgtgacaaaataaacttattccgt ggtgtctttgcgtttcttttatatgttgccacctt tatgtatgtattttctacgtttgctaacatactgc gtaataaggagtcttaatcatgccagttcttttgg gtattccgttattattgcgtttcctcggtttcctt ctggtaactttgttcggctatctgcttacttttct taaaaagggcttcggtaagatagctattgctattt cattgtttcttgctcttattattgggcttaactca attcttgtgggttatctctctgatattagcgctca attaccctctgactttgttcagggtgttcagttaa ttctcccgtctaatgcgcttccctgtttttatgtt attctctctgtaaaggctgctattttcatttttga cgttaaacaaaaaatcgtttcttatttggattggg ataaataatatggctgtttattttgtaactggcaa attaggctctggaaagacgctcgttagcgttggta agattcaggataaaattgtagctgggtgcaaaata gcaactaatcttgatttaaggcttcaaaacctccc gcaagtcgggaggttcgctaaaacgcctcgcgttc ttagaataccggataagccttctatatctgatttg cttgctattgggcgcggtaatgattcctacgatga aaataaaaacggcttgcttgttctcgatgagtgcg gtacttggtttaatacccgttcttggaatgataag gaaagacagccgattattgattggtttctacatgc tcgtaaattaggatgggatattatttttcttgttc aggacttatctattgttgataaacaggcgcgttct gcattagctgaacatgttgtttattgtcgtcgtct ggacagaattactttaccttttgtcggtactttat attctcttattactggctcgaaaatgcctctgcct aaattacatgttggcgttgttaaatatggcgattc tcaattaagccctactgttgagcgttggctttata ctggtaagaatttgtataacgcatatgatactaaa caggctttttctagtaattatgattccggtgttta ttcttatttaacgccttatttatcacacggtcggt atttcaaaccattaaatttaggtcagaagatgaaa ttaactaaaatatatttgaaaaagttttctcgcgt tctttgtcttgcgattggatttgcatcagcattta catatagttatataacccaacctaagccggaggtt aaaaaggtagtctctcagacctatgattttgataa attcactattgactcttctcagcgtcttaatctaa gctatcgctatgttttcaaggattctaagggaaaa ttaattaatagcgacgatttacagaagcaaggtta ttcactcacatatattgatttatgtactgtttcca ttaaaaaaggtaattcaaatgaaattgttaaatgt aattaattttgattcttgatgtttgtttcatcatc ttcttttgctcaggtaattgaaatgaataattcgc ctctgcgcgattttgtaacttggtattcaaagcaa tcaggcgaatccgttattgtttctcccgatgtaaa aggtactgttactgtatattcatctgacgttaaac ctgaaaatctacgcaatttctttatttctgtttta cgtgcaaataattttgatatggtaggttctaaccc ttccattattcagaagtataatccaaacaatcagg attatattgatgaattgccatcatctgataatcag gaatatgatgataattccgctccttctggtggttt ctttgttccgcaaaatgataatgttactcaaactt ttaaaattaataacgttcgggcaaaggatttaata cgagttgtcgaattgtttgtaaagtctaatacttc taaatcctcaaatgtattatctattgacggctcta atctattagttgttagtgctcctaaagatatttta gataaccttcctcaattcctttcaactgttgattt gccaactgaccagatattgattgagggtttgatat ttgaggttcagcaaggtgatgctttagatttttca tttgctgctggctctcagcgtggcactgttgcagg cggtgttaatactgaccgcctcacctctgattatc ttctgctggtggttcgttcggtatttttaatggcg atgattagggctatcagttcgcgcattaaagacta atagccattcaaaaatattgtctgtgccacgtatt cttacgctttcaggtcagaagggttctatctctgt tggccagaatgtcccttttattactggtcgtgtga ctggtgaatctgccaatgtaaataatccatttcag acgattgagcgtcaaaatgtaggtatttccatgag cgtttttcctgttgcaatggctggcggtaatattg ttctggatattaccagcaaggccgatagtttgagt tcttctactcaggcaagtgatgttattactaatca aagaagtattgctacaacggttaatttgcgtgatg gacagactcttttactcggtggcctcactgattat aaaaacacttctcaggattctggcgtaccgttcct gtctaaaatccctttaatcggcctcctgtttagct cccgctctgattctaacgaggaaagcacgttatac gtgctcgtcaaagcaaccatagtacgcgccctgta gcggcgcattaagcgcggcgggtgtggtggttacg cgcagcgtgaccgctacacttgccagcgccctagc gcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaat cgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgatttgggtg atggttcacgtagtgggccatcgccctgatagacg gtttttcgccctttgacgttggagtccacgttctt taatagtggactcttgttccaaactggaacaacac tcaaccctatctcgggctattcttttgatttataa gggattttgccgatttcggaaccaccatcaaacag gattttcgcctgctggggcaaaccagcgtggaccg cttgctgcaactctctcagggccaggcggtgaagg gcaatcagctgttgcccgtctcactggtgaaaaga aaaaccaccctggcgcccaatacgcaaaccgcctc tccccgcgcgttggccgattcattaatgcagctgg cacgacaggtttcccgactggaaagcgggcagtga gcgcaacgcaattaatgtgagttagctcactcatt aggcaccccaggctttacactttatgcttccggct cgtatgttgtgtggaattgtgagcggataacaatt tcacacaggaaacagctatgaccatgattacgaat tcgagctctgactactgcttggattctcaccaata aaaaacgcccggcggcaaccgagcgttctgaacaa atccagatggagttctgaggtcattactggatcta tcaacaggagtccaagccaattctcgaacgtttca attcagcggcaacgggttgccgtgataacggctgg caaagaacagcagcggctggggggcattcgcctcc ggcactccacaggcgctaacgcgcgcaatgaagag cgtgtggtcgtgtacttcaaccgtgctctccacct cgcattcgaaccaggccatggcgtcccgcagagtg gggaggccggcctgaactgtgaaagcaggaggagg agtgccatcgatcacacgcttgctgaagaatgccg ataacatcttctgactttcacccaggaggctgacg ccgaagcgtccgccttgagtcagcagctcatgcat acggcccgacttcagggacaccatcacagtcggcg ggtccagactgatggaggtgaagctgttcaccgtc atgccatgcacttcgccctcgtcagtctccgcgct gagaaccgcaattcccgtcgcgaacagtgcaaccg cctggcggaagctggtggagtcgatatccaccacc acatctttttttaacgtcatttgaatcacccagga gttagggctccgctggccttgaccagcggagcaat agcgtcaggccgcaatagtgggtgcgtactggctg caccactgaccgatacgttccgggctggagaagcg atcccactgaagttccggatagttgaagttgtccg tgaactcatcagccatttcacggctctggctcagg atctgaaggaaggtgaggaattccggcggaagttc cgtgaaggcgctcagagtgaagttggtccagcggg tggcgcacagcacgcgatcctggcggcgacgctcc aggtgttcgctgaagcgcaggtcgtagacagagtg cgcaaggatttcctcgcccagaatccatgccgcat aggacgccatgttcgcgccctggcccaagaccgga tcgacagttgcctggatgtcgcccagcccgatgat ggttttgccgttattgagggtcgcatgaccgtcgc ggaatactggcacaacaccgccctggaggatgtcc agagaactgttggccaggtcgaactcagccggatc gatgcgctcggcaacggaaggatgatgcttacgca gcttctccagcatcagatcgaggaacgcacgcggg tcatcgtcatacttggtgtgggcgaggacttccag atcgctaccaatatggttttcgagcaccagcgctg tgctcatgccattgaacgacagggttggaatctca atcagctcgccatgccctggcgagaaggacatagt caccgcgcgaatcggtgcttccttgatgcccttga agagaccaacgcacagtgcccgttgcggcttctcg aagggcgagttttcggactgcttctcgaacacctt gccgagggcgtatttaccagtgcacacaaccagca gatcatactgctccgacagcccttccagatcttcg gcagacacggcgtcgtagcagaacttgccgcccct ggcttccagtgcacgcatcagcatcggcaggtaga gacggtagtccactgcacggctgggagccttgaga tcaccgtagaaacgcatgggctgcggcccacctac gtagtagtagtggccgaaatagccaaactcctcag acggccactcattgacgtcgagggcaacctcccgc tgcaccgtcaccgcgttgtgagcaacggtattcag gagccgcagtccactgtactcatcgggcttacgat cagtgtacacagtgacgtcgacgtcatgctggcgg aggaagaggccaagatggaggccggcagtgcctgc accaacaataccgatacgctttttcatgcatgcta cctccttaatccttcagctgggtcagtgcgtcctg ctgatgtgctcagtatctctatcactgatagggat gtcaatctctatcactgatagggaggtacccgggg atcctctagagtcgacctgcaggcatgcaagcttg gcactggccgtcgttttacaacgtcgtgactggga aaaccctggcgttacccaacttaatcgccttgcag cacatccccctttcgccagctggcgtaatagcgaa gaggcccgcaccgatcgcccttcccaacagttgcg cagcctgaatggcgaatggcgctttgcctggtttc cggcaccagaagcggtgccggaaagctggctggag tgcgatcttcctgaggccgatactgtcgtcgtccc ctcaaactggcagatgcacggttacgatgcgccca tctacaccaacgtgacctatcccattacggtcaat ccgccgtttgttcccacggagaatccgacgggttg ttactcgctcacatttaatgttgatgaaagctggc tacaggaaggccagacgcgaattatttttgatggc gttcctattggttaaaaaatgagctgatttaacaa aaatttaatgcgaattttaacaaaatattaacgtt tacaatttaaatatttgcttatacaatcttcctga tttggggcttttctgattatcaaccggggtacata tgattgacatgctagttttacgattaccgttcatc gattctcttgtttgctccagactctcaggcaatga cctgatagcctttgtagatctctcaaaaatagcta ccctctccggcattaatttatcagctagaacggtt gaatatcatattgatggtgatttgactgtctccgg cctttctcacccttttgaatctttacctacacatt actcaggcattgcatttaaaatatatgagggttct aaaaatttttatccttgcgttgaaataaaggcttc tcccgcaaaagtattacagggtcataatgtttttg gtacaaccgatttagctttatgctctgaggcttta ttgcttaattttgctaattctttgccttgcctgta tgatttattggatgtt (pLT006) SEQ ID NO: 7 gacgtctgtgcaagtactactgttctgcagtcact tgaattcgatacccagctgggtggagtgcaccaag gagcatgcgaaggaaacgtttcgcagaagcttccg caaggtaccactttgccgcggagtatttgtacatt tgaaggatcctcaagtcggccgcccgttccatgga tactcgtcgaccattacgctagccgtctggagctc ggactgcttaagtcgctccatatgctgaaatgagc tgttgacaattaatcatccggctcgtataatgtgt ggaattgtgagcggataacaatttcacaccccggg ataaggaggacaattgatgcgtaaaggagaagaac ttttcactggagttgtcccaattcttgttgaatta gatggtgatgttaatgggcacaaattttctgtcag tggagagggtgaaggtgatgcaacatacggaaaac ttacccttaaatttatttgcactactggaaaacta cctgttccgtggccaacacttgtcactactttcgg ttatggtgttcaatgctttgcgagatacccagatc acatgaaacagcatgactttttcaagagtgccatg cccgaaggttacgtacaggaaagaactatattttt caaagatgacgggaactacaagacacgtgctgaag tcaagtttgaaggtgatacccttgttaatagaatc gagttaaaaggtattgattttaaagaagatggaaa cattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaag aatggaatcaaagttaacttcaaaattagacacaa cattgaagatggaagcgttcaactagcagaccatt atcaacaaaatactccgattggcgatggccctgtc cttttaccagacaaccattacctgtccacacaatc tgccctttcgaaagatcccaacgaaaagagagacc acatggtccttcttgagtttgtaaccgctgctggg attacacatggcatggatgaactatacaaataagg cgccagggttgatatctatcgccctagggaccgtc tcgagagaatcaatattaatccaacgcgtggcatc aaataaaacgaaaggctcagtcgaaagactgggcc tttcgttttatctgttgtttgtcggtgaacgctct cctgagtaggacaaatccgccgccctagacttagg cgttcggctgcggcgagcggtatcagctcactcaa aggcggtaatacggttatccacagaatcaggggat aacgcaggaaagaacatgtgagcaaaaggccagca aaaggccaggaaccgtaaaaaggccgcgttgctgg cgtttttccataggctccgcccccctgacgagcat cacaaaaatcgacgctcaagtcagaggtggcgaaa cccgacaggactataaagataccaggcgtttcccc ctggaagctccctcgtgcgctctcctgttccgacc ctgccgcttaccggatacctgtccgcctttctccc ttcgggaagcgtggcgctttctcatagctcacgct gtaggtatctcagttcggtgtaggtcgttcgctcc aagctgggctgtctgcacgaaccccccgttcagcc cgaccgctgcgccttatccggtaactatcgtcttg agtccaacccggtaagacacgacttatcgccactg gcagcagccactggtaacaggattagcagagcgag gtatgtaggcggtgctacagagttcttgaagtggt ggcctaactacggctacactagaaggacagtattt ggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaa ccaccgctggtagcggtggttatttgtttgcaagc agcagattacgcgcagaaaaaaaggatctcaagaa gatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtca tggctagtgcttggattctcaccaataaaaaacgc ccggcggcaaccgagcgttctgaacaaatccagat ggagttctgaggtcattactggatctatcaacagg agtccaagccaattctcgaaccccagagtcccgct cagaagaactcgtcaagaaggcgatagaaggcgat gcgctgcgaatcgggagcggcgataccgtaaagca cgaggaagcggtcagcccattcgccgccaagctct tcagcaatatcacgggtagccaacgctatgtcctg atagcggtccgccacacccagccggccacagtcga tgaatccagaaaagcggccattttccaccatgata ttcggcaagcaggcatcgccgtgggtcacgacgag atcctcgccgtcgggcatacgcgccttgagcctgg cgaacagttcggctggcgcgagcccctgatgctct tcgtccagatcatcctgatcgacaagaccggcttc catccgagtacgtgctcgctcgatgcgatgtttcg cttggtggtcgaatgggcaggtagccggatcaagc gtatgcagccgccgcattgcatcagccatgatgga tactttctcggcaggagcaaggtgagatgacagga gatcctgccccggcacttcgcccaatagcagccag tcccttcccgcttcagtgacaacgtcgagcacagc cgcgcaaggaacgcccgtcgtggccagccacgata gccgcgctgcctcgtcttgcagttcattcagggca ccggacaggtcggtcttgacaaaaagaaccgggcg accctgcgctgacagccggaacacggcggcatcag agcagccgattgtctgttgtgcccagtcatagccg aatagcctctccacccaagccgccggagaacctgc gtgcaatccatcttgttcaatcatgcgaaacgatc ctcatcctgtctcttgatcagatcttgatcccctg cgccatcagatccttggcggcaagaaagccatcca gtttactttgcagggcttcccaaccttaccagagg gcggcccaactggcaattcc (pPh006) SEQ ID NO: 8 gacgtctgtgcaagtactactgttctgcagtcact tgaattcgatacccagctgggtggagtgcaccaag gagcatgcgaaggaaacgtttcgcagaagcttccg caaggtaccactttgccgcggagtatttgtacatt tgaaggatcctcaagtcggccgcccgttccatgga tactcgtcgaccattacgctagccgtctggagctc ggactgcttaagtcgctccatatgctgaaatgagc tgttgacaattaatcatccggctcgtataatgtgt ggaattgtgagcggataacaatttcacaccccggg ataaggaggacaattgatgcgtaaaggagaagaac ttttcactggagttgtcccaattcttgttgaatta gatggtgatgttaatgggcacaaattttctgtcag tggagagggtgaaggtgatgcaacatacggaaaac ttacccttaaatttatttgcactactggaaaacta cctgttccgtggccaacacttgtcactactttcgg ttatggtgttcaatgctttgcgagatacccagatc acatgaaacagcatgactttttcaagagtgccatg cccgaaggttacgtacaggaaagaactatattttt caaagatgacgggaactacaagacacgtgctgaag tcaagtttgaaggtgatacccttgttaatagaatc gagttaaaaggtattgattttaaagaagatggaaa cattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaag aatggaatcaaagttaacttcaaaattagacacaa cattgaagatggaagcgttcaactagcagaccatt atcaacaaaatactccgattggcgatggccctgtc cttttaccagacaaccattacctgtccacacaatc tgccctttcgaaagatcccaacgaaaagagagacc acatggtccttcttgagtttgtaaccgctgctggg attacacatggcatggatgaactatacaaataagg cgccagggttgatatctatcgccctagggaccgtc tcgagagcgccctgtagcggcgcattaagcgcggc gggtgtggtggttacgcgcagcgtgaccgctacac ttgccagcgccctagcgcccgctcctttcgctttc ttcccttcctttctcgccacgttcgccggctttcc ccgtcaagctctaaatcgggggctccctttagggt tccgatttagtgctttacggcacctcgaccccaaa aaacttgattagggtgatggttcacgtagtgggcc atcgccctgatagacggtattcgccctttgacgtt ggagtccacgttctttaatagtggactcttgttcc aaactggaacaacactcaaccctatctcggtctat tcttttgatttataagggattttgccgatttcggc ctattggttaaaaaatgagctgatttaacaaaaat ttaacgcgaattaaatattaatccaacgcgtggca tcaaataaaacgaaaggctcagtcgaaagactggg cctttcgttttatctgttgtttgtcggtgaacgct ctcctgagtaggacaaatccgccgccctagactta ggcgttcggctgcggcgagcggtatcagctcactc aaaggcggtaatacggttatccacagaatcagggg ataacgcaggaaagaacatgtgagcaaaaggccag caaaaggccaggaaccgtaaaaaggccgcgttgct ggcgtttttccataggctccgcccccctgacgagc atcacaaaaatcgacgctcaagtcagaggtggcga aacccgacaggactataaagataccaggcgtttcc ccctggaagctccctcgtgcgctctcctgttccga ccctgccgcttaccggatacctgtccgcctttctc ccttcgggaagcgtggcgctttctcatagctcacg ctgtaggtatctcagttcggtgtaggtcgttcgct ccaagctgggctgtctgcacgaaccccccgttcag cccgaccgctgcgccttatccggtaactatcgtct tgagtccaacccggtaagacacgacttatcgccac tggcagcagccactggtaacaggattagcagagcg aggtatgtaggcggtgctacagagttcttgaagtg gtggcctaactacggctacactagaaggacagtat ttggtatctgcgctctgctgaagccagttaccttc ggaaaaagagttggtagctcttgatccggcaaaca aaccaccgctggtagcggtggtttttttgtttgca agcagcagattacgcgcagaaaaaaaggatctcaa gaagatcctttgatcttttctacggggtctgacgc tcagtggaacgaaaactcacgttaagggattttgg tcatggctagtgcttggattctcaccaataaaaaa cgcccggcggcaaccgagcgttctgaacaaatcca gatggagttctgaggtcattactggatctatcaac aggagtccaagccaattctcgaaccccagagtccc gctcagaagaactcgtcaagaaggcgatagaaggc gatgcgctgcgaatcgggagcggcgataccgtaaa gcacgaggaagcggtcagcccattcgccgccaagc tcttcagcaatatcacgggtagccaacgctatgtc ctgatagcggtccgccacacccagccggccacagt cgatgaatccagaaaagcggccattttccaccatg atattcggcaagcaggcatcgccgtgggtcacgac gagatcctcgccgtcgggcatacgcgccttgagcc tggcgaacagttcggctggcgcgagcccctgatgc tcttcgtccagatcatcctgatcgacaagaccggc ttccatccgagtacgtgctcgctcgatgcgatgtt tcgcttggtggtcgaatgggcaggtagccggatca agcgtatgcagccgccgcattgcatcagccatgat ggatactttctcggcaggagcaaggtgagatgaca ggagatcctgccccggcacttcgcccaatagcagc cagtcccttcccgcttcagtgacaacgtcgagcac agccgcgcaaggaacgcccgtcgtggccagccacg atagccgcgctgcctcgtcttgcagttcattcagg gcaccggacaggtcggtcttgacaaaaagaaccgg gcgaccctgcgctgacagccggaacacggcggcat cagagcagccgattgtctgttgtgcccagtcatag ccgaatagcctctccacccaagccgccggagaacc tgcgtgcaatccatcttgttcaatcatgcgaaacg atcctcatcctgtctcttgatcagatcttgatccc ctgcgccatcagatccttggcggcaagaaagccat ccagtttactttgcagggcttcccaaccttaccag agggcggcccaactggcaattcc (M6G bacteriophage) SEQ ID NO: 9 aatgctactactattagtagaattgatgccacctt ttcagctcgcgccccaaatgaaaatatagctaaac aggttattgaccatttgcgaaatgtatctaatggt caaactaaatctactcgttcgcagaattgggaatc aactgttatatggaatgaaacttccagacaccgta ctttagttgcatatttaaaacatgttgagctacag cattatattcagcaattaagctctaagccatccgc aaaaatgacctcttatcaaaaggagcaattaaagg tactctctaatcctgacctgttggagtttgcttcc ggtctggttcgctttgaagctcgaattaaaacgcg atatttgaagtctttcgggcttcctcttaatcttt ttgatgcaatccgctttgcttctgactataatagt cagggtaaagacctgatttttgatttatggtcatt ctcgttttctgaactgtttaaagcatttgaggggg attcaatgaatatttatgacgattccgcagtattg gacgctatccagtctaaacattttactattacccc ctctggcaaaacttcttttgcaaaagcctctcgct attttggtttttatcgtcgtctggtaaacgagggt tatgatagtgttgctcttactatgcctcgtaattc cttttggcgttatgtatctgcattagttgaatgtg gtattcctaaatctcaactgatgaatctttctacc tgtaataatgttgttccgttagttcgttttattaa cgtagatttttcttcccaacgtcctgactggtata atgagccagttcttaaaatcgcataaggtaattca caatgattaaagttgaaattaaaccatctcaagcc caatttactactcgttctggtgtttctcgtcaggg caagccttattcactgaatgagcagctttgttacg ttgatttgggtaatgaatatccggttcttgtcaag attactcttgatgaaggtcagccagcctatgcgcc tggtctgtacaccgttcatctgtcctctttcaaag ttggtcagttcggttcccttatgattgaccgtctg cgcctcgttccggctaagtaacatggagcaggtcg cggatttcgacacaatttatcaggcgatgatacaa atctccgttgtactttgtttcgcgcttggtataat cgctgggggtcaaagatgagtgttttagtgtattc ttttgcctctttcgttttaggttggtgccttcgta gtggcattacgtattttacccgtttaatggaaact tcctcatgaaaaagtctttagtcctcaaagcctct gtagccgttgctaccctcgttccgatgctgtcttt cgctgctgagggtgacgatcccgcaaaagcggcct ttaactccctgcaagcctcagcgaccgaatatatc ggttatgcgtgggcgatggttgttgtcattgtcgg cgcaactatcggtatcaagctgtttaagaaattca cctcgaaagcaagctgataaaccgatacaattaaa ggctccttttggagccttttttttggagattttca acgtgaaaaaattattattcgcaattcctttagtt gttcctttctattctcactccgctgaaactgttga aagttgtttagcaaaatcccatacagaaaattcat ttactaacgtctggaaagacgacaaaactttagat cgttacgctaactatgagggctgtctgtggaatgc tacaggcgttgtagtttgtactggtgacgaaactc agtgttacggtacatgggttcctattgggcttgct atccctgaaaatgagggtggtggctctgagggtgg cggttctgagggtggcggttctgagggtggcggta ctaaacctcctgagtacggtgatacacctattccg ggctatacttatatcaaccctctcgacggcactta tccgcctggtactgagcaaaaccccgctaatccta atccttctcttgaggagtctcagcctcttaatact ttcatgtttcagaataataggttccgaaataggca gggggcattaactgtttatacgggcactgttactc aaggcactgaccccgttaaaacttattaccagtac actcctgtatcatcaaaagccatgtatgacgctta ctggaacggtaaattcagagactgcgctttccatt ctggctttaatgaggatttatttgtttgtgaatat caaggccaatcgtctgacctgcctcaacctcctgt caatgctggcggcggctctggtggtggttctggtg gcggctctgagggtggtggctctgagggtggcggt tctgagggtggcggctctgagggaggcggttccgg tggtggctctggttccggtgattttgattatgaaa agatggcaaacgctaataagggggctatgaccgaa aatgccgatgaaaacgcgctacagtctgacgctaa aggcaaacttgattctgtcgctactgattacggtg ctgctatcgatggtttcattggtgacgtttccggc cttgctaatggtaatggtgctactggtgattttgc tggctctaattcccaaatggctcaagtcggtgacg gtgataattcacctttaatgaataatttccgtcaa tatttaccttccctccctcaatcggttgaatgtcg cccttttgtctttggcgctggtaaaccatatgaat tttctattgattgtgacaaaataaacttattccgt ggtgtctttgcgtttcttttatatgttgccacctt tatgtatgtattttctacgtttgctaacatactgc gtaataaggagtcttaatcatgccagttcttttgg gtattccgttattattgcgtttcctcggtttcctt ctggtaactttgttcggctatctgcttacttttct taaaaagggcttcggtaagatagctattgctattt cattgtttcttgctcttattattgggcttaactca attcttgtgggttatctctctgatattagcgctca attaccctctgactttgttcagggtgttcagttaa ttctcccgtctaatgcgcttccctgtttttatgtt attctctctgtaaaggctgctattttcatttttga cgttaaacaaaaaatcgtttcttatttggattggg ataaataatatggctgtttattttgtaactggcaa attaggctctggaaagacgctcgttagcgttggta agattcaggataaaattgtagctgggtgcaaaata gcaactaatcttgatttaaggcttcaaaacctccc gcaagtcgggaggttcgctaaaacgcctcgcgttc ttagaataccggataagccttctatatctgatttg cttgctattgggcgcggtaatgattcctacgatga aaataaaaacggcttgcttgttctcgatgagtgcg gtacttggtttaatacccgttcttggaatgataag gaaagacagccgattattgattggtttctacatgc tcgtaaattaggatgggatattatttttcttgttc aggacttatctattgttgataaacaggcgcgttct gcattagctgaacatgttgtttattgtcgtcgtct ggacagaattactttaccttttgtcggtactttat attctcttattactggctcgaaaatgcctctgcct aaattacatgttggcgttgttaaatatggcgattc tcaattaagccctactgttgagcgttggctttata ctggtaagaatttgtataacgcatatgatactaaa caggctttttctagtaattatgattccggtgttta ttcttatttaacgccttatttatcacacggtcggt atttcaaaccattaaatttaggtcagaagatgaaa ttaactaaaatatatttgaaaaagttttctcgcgt tctttgtcttgcgattggatttgcatcagcattta catatagttatataacccaacctaagccggaggtt aaaaaggtagtctctcagacctatgattttgataa attcactattgactcttctcagcgtcttaatctaa gctatcgctatgttttcaaggattctaagggaaaa ttaattaatagcgacgatttacagaagcaaggtta ttcactcacatatattgatttatgtactgtttcca ttaaaaaaggtaattcaaatgaaattgttaaatgt aattaattttgattcttgatgtttgtttcatcatc ttcttttgctcaggtaattgaaatgaataattcgc ctctgcgcgattttgtaacttggtattcaaagcaa tcaggcgaatccgttattgtttctcccgatgtaaa aggtactgttactgtatattcatctgacgttaaac ctgaaaatctacgcaatttctttatttctgtttta cgtgcaaataattttgatatggtaggttctaaccc ttccattattcagaagtataatccaaacaatcagg attatattgatgaattgccatcatctgataatcag gaatatgatgataattccgctccttctggtggttt ctttgttccgcaaaatgataatgttactcaaactt ttaaaattaataacgttcgggcaaaggatttaata cgagttgtcgaattgtttgtaaagtctaatacttc taaatcctcaaatgtattatctattgacggctcta atctattagttgttagtgctcctaaagatatttta gataaccttcctcaattcctttcaactgttgattt gccaactgaccagatattgattgagggtttgatat ttgaggttcagcaaggtgatgctttagatttttca tttgctgctggctctcagcgtggcactgttgcagg cggtgttaatactgaccgcctcacctctgattatc ttctgctggtggttcgttcggtatttttaatggcg atgattagggctatcagttcgcgcattaaagacta atagccattcaaaaatattgtctgtgccacgtatt cttacgctttcaggtcagaagggttctatctctgt tggccagaatgtcccttttattactggtcgtgtga ctggtgaatctgccaatgtaaataatccatttcag acgattgagcgtcaaaatgtaggtatttccatgag cgtttttcctgttgcaatggctggcggtaatattg ttctggatattaccagcaaggccgatagtttgagt tcttctactcaggcaagtgatgttattactaatca aagaagtattgctacaacggttaatttgcgtgatg gacagactcttttactcggtggcctcactgattat aaaaacacttctcaggattctggcgtaccgttcct gtctaaaatccctttaatcggcctcctgtttagct cccgctctgattctaacgaggaaagcacgttatac gtgctcgtcaaagcaaccatagtacgcgccctgta gcggcgcattaagcgcggcgggtgtggtggttacg cgcagcgtgaccgctacacttgccagcgccctagc gcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaat cgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgatttgggtg atggttcacgtagtgggccatcgccctgatagacg gtttttcgccctttgacgttggagtccacgttctt taatagtggactcttgttccaaactggaacaacac tcaaccctatctcgggctattcttttgatttataa gggattttgccgatttcgggactgcttaagtcgct ccatatgctgaaatgagctgttgacaattaatcat ccggctcgtataatgtgtggaattgtgagcggata acaatttcacaccccgggataaggaggacaattga tgcgtaaaggagaagaacttttcactggagttgtc ccaattcttgttgaattagatggtgatgttaatgg gcacaaattttctgtcagtggagagggtgaaggtg atgcaacatacggaaaacttacccttaaatttatt tgcactactggaaaactacctgttccgtggccaac acttgtcactactttcggttatggtgttcaatgct ttgcgagatacccagatcacatgaaacagcatgac tttttcaagagtgccatgcccgaaggttacgtaca ggaaagaactatatttttcaaagatgacgggaact acaagacacgtgctgaagtcaagtttgaaggtgat acccttgttaatagaatcgagttaaaaggtattga ttttaaagaagatggaaacattcttggacacaaat tggaatacaactataactcacacaatgtatacatc atggcagacaaacaaaagaatggaatcaaagttaa cttcaaaattagacacaacattgaagatggaagcg ttcaactagcagaccattatcaacaaaatactccg attggcgatggccctgtccttttaccagacaacca ttacctgtccacacaatctgccctttcgaaagatc ccaacgaaaagagagaccacatggtccttcttgag tttgtaaccgctgctgggattacacatggcatgga tgaactatacaaataaggcgccagggttgatatct atcgccctagggaccgtctcgagagaatcaatatt aatccaacgcgtggcatcaaataaaacgaaaggct cagtcgaaagactgggcctttcgttttatctgttg tttgtcggtgaacgctctcctgagtaggacaaatc cgccgccctagactattggttaaaaaatgagctga tttaacaaaaatttaatgcgaattttaacaaaata ttaacgtttacaatttaaatatttgcttatacaat cttcctgtttttggggcttttctgattatcaaccg gggtacatatgattgacatgctagttttacgatta ccgttcatcgattctcttgtttgctccagactctc aggcaatgacctgatagcctttgtagatctctcaa aaatagctaccctctccggcattaatttatcagct agaacggttgaatatcatattgatggtgatttgac tgtctccggcctttctcacccttttgaatctttac ctacacattactcaggcattgcatttaaaatatat gagggttctaaaaatttttatccttgcgttgaaat aaaggcttctcccgcaaaagtattacagggtcata atgtttttggtacaaccgatttagctttatgctct gaggctttattgcttaattttgctaattctttgcc ttgcctgtatgatttattggatgtt

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.

This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. 

1.-21. (canceled)
 22. A recombinant bacteriophage that is engineered for in vivo delivery to endogenous bacterial cells to contain at least one genetic circuit, wherein the at least one genetic circuit does not express an antimicrobial protein.
 23. The recombinant bacteriophage of claim 22, wherein the recombinant bacteriophage is a non-lytic recombinant bacteriophage.
 24. The recombinant bacteriophage of claim 23, wherein the non-lytic recombinant bacteriophage is of a family selected from Myoviridae, Siphoviridae, Podoviridae, Tectiviridae, Corticoviridae, Lipothrixviridae, Plasmaviridae, Rudiviridae, Fuselloviridae, Inoviridae, Microviridae, Leviviridae and Cystoviridae.
 25. The recombinant bacteriophage of claim 24, wherein the non-lytic recombinant bacteriophage is an Inoviridae M13 or M13-like bacteriophage.
 26. The recombinant bacteriophage of claim 22, wherein the at least one genetic circuit contains a nucleic acid with a promoter operably linked to a nucleotide sequence encoding a gene product.
 27. The recombinant bacteriophage of claim 26, wherein the nucleic acid is a recombinant nucleic acid.
 28. The recombinant bacteriophage of claim 26, wherein the nucleic acid is a synthetic nucleic acid.
 29. The recombinant bacteriophage of claim 26, wherein the promoter is a constitutively active promoter.
 30. The recombinant bacteriophage of claim 26, wherein the promoter is an inducible promoter.
 31. The recombinant bacteriophage of claim 26, wherein the at least one genetic circuit is a recombinase-based genetic circuit.
 32. The recombinant bacteriophage of claim 22, wherein the at least one genetic circuit is engineered to express a therapeutic molecule.
 33. The recombinant bacteriophage of claim 32, wherein the at least one genetic circuit comprises a nucleic acid with a promoter operably linked to a nucleotide sequence that encodes the therapeutic molecule.
 34. The recombinant bacteriophage of claim 32, wherein the therapeutic molecule is an antibody, antibody-based drug, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin or thrombolytic.
 35. The recombinant bacteriophage of claim 22, wherein the at least one genetic circuit is engineered to detect a condition.
 36. The recombinant bacteriophage of claim 35, wherein the at least one genetic circuit comprises a nucleic acid with an inducible promoter operably linked to a nucleotide sequence that encodes a reporter molecule.
 37. The recombinant bacteriophage of claim 35, wherein the condition is a cancer, an immune disorder or an infection.
 38. The recombinant bacteriophage of claim 36, wherein the reporter molecule is fluorescent protein.
 39. The recombinant bacteriophage of claim 22, wherein the at least one genetic circuit comprises a nucleic acid with a promoter operably linked to a nucleotide sequence that encodes toluene dioxygenase or styrene monoxygenase.
 40. A composition comprising the recombinant bacteriophage of claim
 22. 41.-76. (canceled) 